Journal: bioRxiv

Article Title: Distinct non-coding RNA cargo of extracellular vesicles from M1 and M2 human primary macrophages

doi: 10.1101/2022.08.19.504493

Figure Lengend Snippet: Extracellular vesicles were isolated by size exclusion chromatography (SEC) and characterised by nanoparticle tracking analysis (NTA), electron microscopy, western blotting, and ELISA. a) Representative EV count measured by NTA (solid blue line; right Y axis) in the first eight SEC fractions overlaid with the protein concentration in 26 SEC fractions (dashed orange line; left Y axis) showing separation of EVs from soluble protein. b) Representative image of silver staining for the 26 SEC fractions. c) Total particle count measured by NTA in fractions 1-3, per million PBMC seeded. Wilcoxon matched-pairs signed rank test, p≤0.01, n=12. d) Median size of EVs from M1 and M2 cells, measured by NTA. Wilcoxon matched-pairs signed rank test, p≥0.01, n=12. e) Representative size profile of M1 (red) and M2 (blue) macrophage EVs measured by NTA. f) Transmission electron micrographs of EVs from M1 and M2 macrophages. g) ELISA for the canonical EV surface markers CD63 and HLA-A in the first five SEC fractions. h) Western blot for the luminal EV marker GAPDH and the negative EV marker Calnexin.

Article Snippet: Gene expression was determined using TaqMan Gene Expression Assays containing a FAM dye labelled probe (Applied Biosystems) duplexed with VIC dye labelled probe against GAPDH (Hs99999905_m1, Applied Biosystems) as housekeeping expression control.

Techniques: Isolation, Size-exclusion Chromatography, Electron Microscopy, Western Blot, Enzyme-linked Immunosorbent Assay, Protein Concentration, Silver Staining, Transmission Assay, Marker

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: (A) Schematic of cell-free protein expression into synthetic lipid vesicles and subsequent electroformation with Vesicle Prep Pro (Nanion). ( B ) Full-length PKD2L1-GFP protein detected by western blot after cell-free expression into vesicles. ( C ) Monitored fluorescence over time of cell-free expressed PKD2L1-GFP and a non-fluorescent control plasmid produced in the presence or absence of lipid vesicles. Figure 1—source data 1. Original file for western blot analysis displayed in . Figure 1—source data 2. PDF file containing original western blot for , indicating the relevant bands and treatments.

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Expressing, Western Blot, Fluorescence, Control, Plasmid Preparation, Produced

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: (A) Top , fluorescence (488γ) standard curve determined with a recombinant GFP-tagged protein fit with a linear regression. PURExpress synthesized PKD2L1-GFP and PKD2-GFP were measured after 3 hr of expression at 37°C. (GFP standard curve N = 5, PKD2L1 and PKD2 N = 3 replicates). Bottom , average protomer and tetramer protein production from the PURExpress reaction. Error bars represent SEM. ( B ) Representative sequence coverage of PKD2L1-GFP (top) and PKD2-GFP (bottom) from tandem mass spectrometry spectra. ( C ) Mass spectrometry outputs identifying polycystin proteins. ( D, E ) Fluorescence-detection size-exclusion chromatography (FSEC) of polycystin proteins derived from recombinant and cell-free protein expression (CFE) sources. Recombinant human PKD2-GFP and PKD2L1-GFP protein was obtained from lysates of 0.5 × 10 6 HEK cells stably expressing the channels. Purified Aequorea Victoria GFP His-tag protein was obtained from Thermo Fisher Scientific. SUVs containing CFE-derived polycystins were lysed using dodecyl β- D -maltoside (DDM) prior to FSEC analysis (see methods).

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Fluorescence, Recombinant, Synthesized, Expressing, Sequencing, Mass Spectrometry, Size-exclusion Chromatography, Derivative Assay, Stable Transfection, Purification

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: Schematic of PKD2L1-SNAP incorporated into GUVs, followed by SNAP staining with cell permeable (Cell488), and cell impermeable (Surface647) SNAP-Tag marker.

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Staining, Marker

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: (A) Schematic of possible ion channel orientation outcomes from PKD2L1 cell-free expression (top) and hypothesized fluorescence results when Cell488 and Surface647 added (bottom). ( B ) Fluorescent confocal images from the SNAP-tagged vesicles. The scale bar represents 10 μm for all images. ( C ) Vesicle percentage depicts the percent of vesicles with each fluorescent output ( N = 65 vesicles).

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Expressing, Fluorescence

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: (A) Images of voltage-clamped giant unilamellar vesicles (GUVs) with incorporated PKD2L1-GFP channels. Left , establishing high-resistance seals in the on-cell patch configuration. Right , transitioning to the inside-our patch configuration. Scale bar = 20 μm. ( B ) Example unitary single-channel current records from GUVs reconstituted with or without PKD2L1 protein. Vesicles were patched using the symmetrical 150 mM K + conditions (see methods) and PKD2L1 single-channel current producing full and sub-conductances are colored black and blue, respectively. ( C ) Average single-channel current amplitudes. Conductance ( γ ) estimated by fitting the average single-channel currents to a linear equation. Error (gray) indicates SEM from N = 3–8 GUVs. Several replicates lacked single-channel openings at all test potentials. ( D ) PKD2L1 single-channel current recorded using asymmetric cationic solutions, with 150 mM K + in the bath and 150 mM NMDG + in the pipette. ( E ) Resulting average single-channel current amplitudes where no inward single-channel current was detected ( N = 3–4 GUVs).

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Transferring

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: Conductance properties of polycystins measured from giant unilamellar vesicle (GUV) and cilia membranes.

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Membrane, Expressing

Journal: eLife

Article Title: A synthetic method to assay polycystin channel biophysics

doi: 10.7554/eLife.98534

Figure Lengend Snippet: ( A, B ) Top , images of voltage-clamped primary cilia from mouse hippocampal neurons and inner medullary kidney collecting duct cells (IMCD) harvested from transgenic mice expressing a fluorescent cilia reporter (ARL13B-EGFP tg ) . Scale bar = 20 μm. Previous work has genetically identified PKD2L1 and PKD2 as essential ion channel subunit in the primary cilia of the renal collecting duct cells and hippocampal neurons ( ; ). Bottom , average single-channel current amplitudes recorded from primary cilia using K + in the recording electrode solution. Conductance ( γ ) estimated by fitting the average single-channel currents to a linear equation. Error indicates SEM ( N = 6 cilia).

Article Snippet: Plasmid DNA encoding human PKD2L1 with C-terminally tagged green fluorescent protein (PKD2L1-GFP) was added to the cell-free expression components (PURExpress, New England Biolabs) and induced protein translation by heat ( , see methods).

Techniques: Transgenic Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Carboxyl-terminal Tail-mediated Homodimerizations of Sphingomyelin Synthases Are Responsible for Efficient Export from the Endoplasmic Reticulum *

doi: 10.1074/jbc.M116.746602

Figure Lengend Snippet: The two N-terminal tails, the N- and C-terminal tails, and the two C-terminal tails in the SMS homodimers are in close proximity. A, schematic of the chimeric proteins used for BiFC assays. The chimeras included VN (N-terminal residues 1–173 of the Venus protein) and V5 epitope or VC (C-terminal residues 155–238 of the Venus protein) and FLAG epitope. B–D, COS7 cells were transfected with a plasmid encoding V5-tagged SMS2-VN and FLAG-tagged SMS2-VC or V5-tagged SMS2-VN and FLAG-tagged VC-GlcT. 24 h post-transfection, the cells were fixed and permeabilized with 0.1% Triton X-100. For confocal microscopy (B), the cells were stained with anti-V5 and anti-FLAG antibodies, followed by appropriate Alexa Fluor-conjugated secondary antibodies. For flow cytometry (C and D), the cells were stained with anti-V5 antibody, followed by PerCP/Cy5.5-conjugated secondary antibody and APC-conjugated anti-FLAG antibody. B, SMS2-VN, blue; SMS2-VC or VC-GlcT, red; Venus, green. B, a close-up view of the plasma membrane in the squared region in a. Scale bar = 10 μm. C, representative dot plot from cells expressing SMS2-VN/SMS2-VC (left panel) and SMS2-VN/VC-GlcT (right panel). The gating of the double-positive cell population expressing both VN- and VC-fused proteins is shown as a bold square. D, FACS histogram representing the intensities of Venus fluorescence detected by the FITC channel in double-positive cells (PerCP-Cy5.5/APC). Histograms represent the extent of FITC quantified in cells expressing SMS2-VN/SMS2-VC (filled) or SMS2-VN/VC-GlcT (open). E and F, the intensity of Venus fluorescence in COS7 cells co-expressing the indicated combinations of VN- or VC-fused SMS1 (E) or SMS2 (F) was quantified by flow cytometry. Individual data points are shown as a scatterplot. Values represent the mean ± S.D. from at least three independent experiments. *, p < 0.01; **, p < 0.05. G and H, schematics of SMS homodimers, viewed as a section through the Golgi or plasma membrane (G) and viewed from the cytosolic space (H). Individual monomers of transmembrane segments in SMSs are shown in black and gray. N- and C-terminal tails are represented by blue and red lines, respectively. 1–6 and I-VI indicated the transmembrane helices. H, SMS1 and SMS2 have the following intermolecular interactions: between the N-terminal tails, between the N-terminal tail and C-terminal tail, and between the C-terminal tails, shown as dashed arrow lines.

Article Snippet: Co-immunoprecipitation was performed with anti-FLAG M2 beads at 4 °C for 4 h. Then the beads were washed four times with lysis buffer and eluted in SDS sample buffer by incubation at 65 °C for 4 min. Co-immunoprecipitated proteins were subjected to SDS-PAGE with Dr. Western (Oriental Yeast) or WIDE-VIEW TM Prestained Protein Size Marker III (Wako).

Techniques: FLAG-tag, Transfection, Plasmid Preparation, Confocal Microscopy, Staining, Flow Cytometry, Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Optimization of pGC-A expression in Sf9 cells. Western blot (anti-pGC-A) of total cell protein over different virus multiplicities of infection (MOI) and transfection time. MOI of 0 indicates no virus transfection. pGC-A (red arrow) is approximately 120 kDa.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Expressing, Western Blot, Infection, Transfection

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Determination of expressed full-length pGC-A functionality via whole-cell activity assay. The competitive ELISA assay measured the cGMP yield level in Sf9 cells expressing full-length pGC-A versus control Sf9 cells (n = 2). Both cell types were incubated with different concentrations of MANP ligand (0 to 10 7 pmol). Incubation with 0 pmol MANP served as a negative control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Activity Assay, Competitive ELISA, Expressing, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purification of full-length pGC-A via affinity and size exclusion columns. ( A ) Western blot (anti-pGC-A) of samples from cell lysis to the affinity column. pGC-A (red arrow) is ~ 120 kDa. ( B ) Coomassie blue stain from cell lysis to the affinity column. ( C ) Coomassie blue stain of the eluted fraction from Superose 6 that was used for protein crystallization. M: marker; P: membrane pellet from ultracentrifugation after solubilization with n-dodecyl-β-D-maltoside (DDM) and cholesteryl hemisuccinate (CHS); FT: flowthrough from the affinity column; 50, 100, 500: imidazole (mM) at elution from the affinity column.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, Western Blot, Lysis, Affinity Column, Staining, Crystallization Assay, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Presence of pGC-A in crystals was confirmed via western blot. The anti-pGC-A antibody was used in the western blot. The pGC-A monomer is 120 kDa. Lane 1: combined mix: combined crystallization drops collected in the PCR tube. Lane 2: supernatant: the supernatant collected from the centrifuged combined mix. Lane 3: washed pellet: the crystal pellet was washed with the precipitant solution and collected again via centrifugation. Lane 4: washed supernatant: the supernatant from washed crystal pellet. Lane 5: crystallization drop: one crystallization hanging drop directly mixed with SDS sample buffer. Lane 6: crystallization sample: purified pGC-A before crystallization, serving as a positive control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Western Blot, Crystallization Assay, Centrifugation, Purification, Positive Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Indexed diffraction patterns from pGC-A microcrystals collected via serial crystallography at the Advanced Photon Source. Three indexable diffraction patterns with the highest resolution of 3 Å. ( A ) Blue/pink diffraction graphs show diffraction patterns analyzed using CrystFEL software. ( B ) The original diffraction patterns shown with white background. All diffraction dots were manually circled in red for better visualization.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Software

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Dynamic oligomeric states of pGC-A seen in replicate runs of Superose 6 size exclusion chromatography may be dependent on protein concentration. ( A ) The Superose 6 10/300GL column performance profile. Five standard proteins were used to generate relative molecule elution points based on different molecular sizes. Thyroglobulin (669 kDa) eluted at 14.19 mL, ferritin (440 kDa) eluted at 15.96 mL, aldolase (158 kDa) eluted at 17.58 mL, ovalbumin (44 kDa) eluted at 18.49 mL, and aprotinin (6.5 kDa) eluted at 21.60 mL. ( B-D ) Size exclusion chromatography of pGC-A. ( B ) The peak intensity at 17.3 mL corresponds to the pGC-A monomeric state (120 kDa). pGC-A monomer is the major peak determined by chromatography. Other ratios were faded out in the background and served as supplemental comparison. ( C ) pGC-A tetramer and monomer present similar ratios in the chromatographic separation. The peak intensity at 14.76 mL and 17.29 mL corresponds to pGC-A tetrameric (480 kDa) and monomeric states, respectively. ( D ) The pGC-A tetramer is the major peak. The peak intensity at 15.05 mL corresponds to the pGC-A tetrameric state.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Size-exclusion Chromatography, Protein Concentration, Chromatography

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stain of high resolution clear-native PAGE from pGC-A Superose 6 fractions. Superose 6 column eluted tetramer (480 kDa) and monomer (120 kDa) size peaks were concentrated and analyzed in a 4–16% native gel. M, marker.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Silver Staining, Clear Native PAGE, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stained clear-native PAGE of different oligomeric samples treated with or without dithiothreitol (DTT) overnight. Three peak samples, which represent the tetramer, dimer, and monomer of full-length pGC-A, were concentrated and split in half for treatment with or without DTT. S, concentrated sample only; S + DTT, concentrated sample incubated with 1 M DTT overnight.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Staining, Clear Native PAGE, Incubation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purified full-length pGC-A in vitro functional activity test. ( A ) Functional activity for two pGC-A oligomeric states with ATP incubation. The control group was GTP and ATP in sample buffer. For activity values in units of mg of purified pGC-A, the y-axis values of pmol/mL can be converted to nmol/mg protein by multiplying by 0.00873. ( B ) Functional activity for two pGC-A oligomeric states without ATP incubation. The control group was GTP only in the sample buffer. ( C ) Competitive cGMP ELISA standard fit in four parameters logistic (4PL) curve. The left Y-axis is the B/B0 (%) value and represents the percentage of bound cGMP. The right Y-axis represents the average net optical density (OD) reading at 405 nm. Both standard curves were generated with a 95% confidence interval. ( D ) The cGMP yield differences between pGC-A oligomer samples incubated with or without ATP were analyzed. All raw data points were analyzed via the ROUT method (Q = 1%) to remove significantly impossible outlier values before data analysis. One-way ANOVA was used to determine the statistical significance between samples and control in graphs ( A ) and ( B ). Two-way ANOVA was used to determine the statistical significance in graph ( D ). Each dot represented to the sample point and plotted as mean ± standard deviation (SD). * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001 and ns P ≥ 0.05, Not significant.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, In Vitro, Functional Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Proposed native state and three-step mechanism of full-length pGC-A. First, the full-length pGC-A forms a tetramer complex in the native state by non-covalent interactions (e.g., hydrogen bond and hydrophobic interactions). In each tetramer complex, there are two functional units, and each functional unit may represent a dimer. The narrowest part of the tetramer is the transmembrane domain. Second, the pGC-A signal transduction mechanism is not ATP-dependent. The current ATP-dependent two-step activation mechanism should instead be three-step. The first step is ligand (ANP) binding, which moderately activates the pGC-A; the second step is binding ATP, which partially boosts protein activity; the third step is the pGC-A phosphorylation, which fully activates the guanylyl cyclase.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Functional Assay, Transduction, Activation Assay, Binding Assay, Activity Assay

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A) The two mutated residues R12 and L68 of SHANK3 are highlighted in red. Both residues are located within the N-terminal SPN domain of SHANK3. (B) The R12C mutation is shown to be localized within an antiparallel beta sheet directly at the SHANK3 N-terminus. (C) The L68P mutation is found in a locally disordered region within the SPN domain. In comparison, the non-mutated L68 residue is located in close vicinity to an antiparallel beta sheet as seen in A. The structures were generated as described in the molecular dynamics section of the materials and methods chapter and were visualized with VMD 1.9.3.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Mutagenesis, Comparison, Residue, Generated

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A) Expression of His 6 -SUMO-SHANK3 (1-676) variants was induced with 300μM IPTG in BL21(DE3) E.coli bacteria and continued for 18 hours at 18°C. Bacteria were lysed and proteins were pre-purified over Ni 2+ - IDA as described in the protein purification section of the materials and methods chapter. For SAXS, proteins were batch eluted from the Ni 2+ - IDA column (bed volume ~2mL) with 200mM Imidazole and were subsequently subjected to size-exclusion chromatography (SEC) using an analytical Superdex 75 10/300 GL column (0.4mL/min; GE Healthcare). Peak fractions were pooled and concentrated to obtain sufficiently pure His 6 -SUMO-SHANK3 (1-676) variants. Due facilitate measurements over a broader concentration range, the His 6 -SUMO-tag was not removed for SAXS studies. (B) For complementary CD spectroscopy and nDSF measurements, the SUMO-tag was removed by treatment with the home-made SUMO protease His 6 -SenP2 (~0.3 mg/mL final concentration) followed by dilution to reduce the Imidazole concentration to approximately 30mM. Removal of the His 6 -SUMO-tag as well as His 6 -SenP2 was done by two additional passages over a freshly packed Ni 2+ - IDA column (bed volume ~2mL). (C) For buffer exchange, diluted proteins were initially concentrated by ultrafiltration and passed over a PD10 desalting column (GE Healthcare). Final protein re-concentration yielded SHANK3 (1-676) variants in sufficient purity for subsequent measurements.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Expressing, Bacteria, Purification, Protein Purification, Size-exclusion Chromatography, Concentration Assay, Circular Dichroism, Buffer Exchange

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A) Size-exclusion chromatograms of Ni 2+ -IDA pre-purified His 6 -SUMO-SHANK3 (1-676) variants are shown (elution peak at ~12.0mL). The individual elution peaks were used for SAXS. (B) R g values derived from Guinier approximation in the range of sR g < 1.3 of SAXS profiles measured at different protein concentrations. The WT and R12C mutant show a linear increase of R g with protein concentration, which suggests the presence of attractive interparticle interactions. (C) Kratky Plots from zero-extrapolated SAXS profiles of the WT and R12C mutant resemble the profile of a folded multidomain protein with flexible linkers, while the L68P mutant appears to be partially unfolded. (D) Distance distribution curves were computed with GNOM using zero-extrapolated SAXS profiles as input data. Particles were assumed to be arbitrary monodisperse. The curves indicate an increased maximum particle diameter (D max ) for both mutants. (E) Rigid-body modelling of the SHANK3 complex topology with CORAL. High-resolution structures of individual SHANK3 fragments were fitted against zero-extrapolated SAXS profiles without assuming any higher order symmetry (space group P1). (F) CORAL models of monomeric His 6 -SUMO-SHANK3 (1-676) indicate distal effects of both mutations on the position of the SH3 domain (orange) and PDZ domain (magenta) relative to the ARR domain (green) as well as changes in the orientation of linker regions.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Purification, Derivative Assay, Mutagenesis, Protein Concentration

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A) Guinier plots show protein concentration dependent changes in the slope of the Guinier fit suggesting the presence of interparticle effects. A suitable Guinier range was detected by the AUTORG function of the ATSAS 3.0.1 software package. (B) PDDFs indicate a concentration dependent increase in the maximum particle diameter (D max ) which corroborates the idea of potential SAM-domain independent self-interaction of SHANK3 (1-676) . All PDDFs were obtained from GNOM analysis using the ATSAS 3.0.1 software package.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Protein Concentration, Software, Concentration Assay

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: For the CORAL input, experimental SAXS profiles have been merged between highest and lowest concentrations. (A) The CORAL fit for His 6 -SUMO-SHANK3 (1-676) WT suggests that the protein could partially exist in a homo-dimeric form in solution, which is formed by a dual interface between SH3 and PDZ domains. The two large ankyrin-rich repeat domains (ARR) are oriented in a cis-conformation as is visible from the front and back view. (B) The ASD-associated R12C mutation is predicted to have no major impact on the complex topology of His 6 -SUMO-SHANK3 (1-676) . (C) The more dominant L68P mutation is predicted to result in an altered orientation of the ARR domains and the position of the SMT3-SPN-ARR domain cluster is altered with respect to the SH3-PDZ cluster.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A) Schematic representation of the SHANK3 (1-676) fragment, which was used for nDSF measurements. Intrinsic tryptophan (Trp) reporter residues are highlighted in the structure, which was derived from SAXS data. (B) SDS-PAGE of Ni 2+ -NTA purified SHANK3 (1-676) variants which were used for nDSF and CD spectroscopy. (C) Overview of detected melting points from peaks of first derivative curves (from E). Due to the complex melting behavior, melting was classified in three transition zones (T m 1 - T m 3) which are partially overlapping. (D) Label-free determination of thermal and chemical stability of purified SHANK3 (1-676) variants by intrinsic fluorescence emission depicted as ratio of 350/330nm as a function of temperature. Melting curves were acquired at a protein concentration of ~0.5mg/mL, 50% excitation power and with a heating rate of 1°C/min. (E) First derivative analysis of melting curves shown in (D). Transition points are indicated with colored arrowheads (color-coded according to the transition zones) and shifted towards lower temperatures with increasing urea concentration, as expected. For the L68P mutant, considerably lower melting points are detected compared to the WT or R12C mutant suggesting a reduced thermal stability of the tertiary structure.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Derivative Assay, SDS Page, Purification, Circular Dichroism, Fluorescence, Protein Concentration, Concentration Assay, Mutagenesis

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: Fluorescence spectra were recorded from 2μM protein solutions at room temperature (excitation wavelength = 295nm, scan speed = 240nm/min). Spectra show a profound reduction in tryptophan fluorescence intensity of the ASD-associated L68P mutant suggesting an increased fraction of exposed tryptophan residues due to solvent quenching, which could be attributed to partial unfolding of the L68P mutant. This effect is absent for the R12C mutant. The inset shows representative SDS gels from Ni 2+ - IDA purified His 6 -SUMO-SHANK3 (1-676) variants.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Fluorescence, Mutagenesis, Solvent, Purification

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A) Isothermal chemical unfolding curves with urea as denaturant are depicted (222nm, 3sec/point, 25μs sample period, 3 repeats, T = 10.0 ± 0.2 °C, protein concentration ~0.15mg/mL). The data has been fitted to a two-state unfolding transition model. Transition midpoints do not vary significantly but differences occur in the slopes of the corresponding folded and unfolded baselines. (B) Unfolding free energies are calculated from fitted parameters obtained in (A) using a linear free energy model. Both mutants show a reduced unfolding cooperativity and thermodynamic stability when unfolding is induced with urea. (C) Thermal unfolding curves have been acquired from samples measured in (B) in the presence of 2.0M Urea to prevent thermally induced protein aggregation preceding actual secondary structure melting (222nm, 3sec/point, 25μs sample period, 10 - 90°C T-range, 1.0°C step size, 1.0°C/min stepped ramping with 30s settling time and 0.5°C tolerance). Thermal unfolding curves reveal an increased secondary structure stability of the R12C mutant (dynode voltage = 370 - 400V between 10 - 90°C). (D) Far-UV CD spectra of SHANK3 (1-676) variants are shown in the range of 260-185nm (0.5nm step size, 3sec/point, 25μs sample period, 3 repeats, T = 10.0 ± 0.2 °C). Immediately before measurement, proteins have been buffer exchanged to 10mM KH 2 PO 4 , 100mM KF, 0.5mM DTT, pH = 6.50. Differences between WT and mutant protein spectra are minor.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Protein Concentration, Mutagenesis, Circular Dichroism

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A) For limited proteolysis at room temperature, SHANK3 (1-676) variants are used at a concentration of 13.5μM (1 mg/mL). As trypsin source, 0.05% (21μM) trypsin-EDTA (Gibco, Thermo Fisher) is pre-diluted with water to 1μM and used in a final concentration of 67nM. For each protein variant, a 8x reaction mix is prepared by mixing SHANK3 (1-676) , pre-diluted trypsin-EDTA and water to obtain a molar ratio of protein:trypsin of 200:1. At each timepoint, an aliquot of the reaction mix is removed and immediately transferred into pre-heated 2x SDS-Loading Buffer at 95°C. Samples are boiled for 5min and subsequently analyzed by SDS-PAGE. (B) Line profile analysis of SDS-PAGE lanes after 1 minute of limited proteolysis of SHANK3 (1-676) variants. Line profiles were measured in Fiji (15px line width) and the molecular weight was estimated based on an exponential decay fit of the running behavior of SDS-PAGE marker bands (PageRuler, 10-180kDa, Thermo Fisher). For the R12C mutant, several fragments show a lower brightness or higher abundance compared to the WT or L68P mutant (region 1 and 3). (C) After 10 minutes of limited proteolysis both mutants show a higher fragment abundance in region 1 compared to the WT. Interestingly, the fragment in region 2 is fully absent for the L68P mutant. (D) After 1 hour of limited proteolysis, both mutants show an increased abundance of residual, uncleaved protein. Additionally, fragments of the R12C mutant in region 1 and 3 are more abundant compared to WT or L68P, consistent with the profile observed after 1 minute. Overall, the R12C mutant seems to exhibit a mildly decreased cleavability which might suggest a higher structural stability or reduced structural accessibility by trypsin. On the other hand, the L68P mutant shows slightly increased cleavage of individual fragments (regions 2 and 3) or is comparable to the profile observed for the WT (region 1) possibly suggesting reduced structural stability or higher accessibility by trypsin.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Concentration Assay, Variant Assay, SDS Page, Molecular Weight, Marker, Mutagenesis

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A) Cα backbone RMSF calculated for the trajectories of the SHANK3 (1-346) fragment (PDB: 5G4X) either as WT or carrying one of the ASD-associated mutations R12C (blue) or L68P (red) show increased fluctuations for both mutants predominantly within the first 100 residues of the protein possibly suggesting a local mutation-induced increase in protein backbone dynamics. (B) Cα backbone RMSD determined for the full protein (aa 1-346). Within the first 100ns both mutants tend to show slightly increased backbone dynamics. This effect inverts with extended sampling time and mutants show on average reduced conformational dynamics compared to the WT. (C) Cα backbone RMSD restricted to the SPN domain of SHANK3 (aa 1-92). The traces resemble those observed for the full protein (aa 1-346) but absolute RMSD values are considerably larger for the SPN domain. (D) Cα backbone RMSD restricted to the ARR domain of SHANK3 (aa 93-346). The WT protein shows a stepwise increase in RMSD over time possibly indicating switches between distinct conformational states. This behavior is largely missing in both mutants but is more prominent for the L68P variant. (E) Local hotspots of conformational dynamics can be seen within the SPN domain (encircled) from overlays of individual frames of the trajectory (every fifth frame loaded, overlay (beginning:step:end) = 0:20:20002 resulting in 1000 frames).

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Mutagenesis, Sampling, Variant Assay

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A) Representative images of immunostained endogenous SHANK3 (red), homer or bassoon (green) and MAP2 (gray). (B) Quantification of the colocalization (SHANK3/Bsn and Bsn/SHANK3: n = 36 cells from two independent cultures; SHANK3/homer and homer/SHANK3: n = 39 cells from two independent cultures). Bars are showing the mean as well as individual data points. ~73% of homer-positive spines are also SHANK3-positive while ~93% of SHANK3-positive spines colocalize with homer. Additionally, ~79% of SHANK3-positive spines colocalize with the pre-synaptic marker bassoon. (C) Quantification of the total number of spines per 10μm in neurons overexpressing GFP-SHANK3 FL variants for 16-18 hours (GFP: 15 cells from 4 independent cultures, WT: 14 cells from 3 independent cultures, R12C: 13 cells from 4 independent cultures, L68P: 13 cells from 4 independent cultures). Bars are showing the mean as well as individual data points. No significant differences in average spine numbers is found (Kruskal-Wallis ANOVA with post-hoc test: p = .59 – 1). (D) Analysis of SHANK3 cluster distribution in neurons overexpressing GFP-SHANK3 FL variants (same dataset as in (C)). Bars are showing mean ± SEM. No significant effect of the R12C mutation on the relative distribution of SHANK3 clusters between spines and dendrites are found. The L68P mutation caused a significant increase in dendritic SHANK3 clusters (Kruskal-Wallis ANOVA with post-hoc test: p(WT / R12C) = .42; p(WT / L68P) = .04 and p(R12C / L68P) = .93 at P=95%). (E) Line-profile analysis of the SHANK3 distribution between spines and dendrites (WT: 12 cells from 4 independent cultures, R12C: 11 cells from 4 independent cultures, L68P: 12 cells from 5 independent cultures). Bars are showing the mean as well as individual data points. No differences in the mean SHANK3 intensity ratio between spines and dendrites are found (one-way ANOVA with Bonferroni multiple comparisons test: p(WT / R12C) = 1 and p(WT / L68P) = .83 at P=95%). (F) FRAP curves showing the dynamics of mutant SHANK3 in spines (same dataset as in (E)). The R12C mutant is displaying an increased recovery corresponding to a reduced residing time in spines (Kruskal-Wallis ANOVA with post-hoc test; untreated: p(WT / R12C) < .0001, p(R12C / L68P) < .0001 and p(WT / L68P) = 1; bic: p(WT / R12C) < .0001, p(R12C / L68P) < .0001 and p(WT / L68P) = 1). All tested SHANK3 variants show a mildly decreased recovery after pharmacological stimulation with (+)-Bicucullin (5min, 37°C, 5% CO 2 ; data collected from 3 independent cultures for control curves and 2 independent cultures for (+)-Bicucullin stimulation). (G) Representative images of selected timepoints during FRAP.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Marker, Mutagenesis, Control

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A-D) In addition to the quantification of spine numbers and cluster densities presented in , representative images of hippocampal primary neurons overexpressing GFP-SHANK3 FL variants for less than 24 hours are shown. Overexpression of ASD-associated mutants does not seem to significantly alter the number and morphology of spines but increases the number of dendritic SHANK3 clusters.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Over Expression

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: (A) To analyze potential changes in global protein turnover of hippocampal primary neurons induced by ASD-associated SHANK3 missense variants, neurons were cultured in the presence of the methionine-analogue L-azidohomoalanine (AHA). Newly synthesized proteins, which incorporated the modified amino acid, were visualized by Cu(I)-catalyzed azide-alkyne click reaction (CuAAC) with tetramethylrhodamine (TAMRA) alkyne. To test the activity-dependence of de novo protein synthesis, neurons were additionally silenced with 1μM tetrodotoxin (TTX) and 50μM D-(-)-2-amino-5-phosphonopentanoic acid (D-APV) prior to metabolic labeling. For each condition representative images (20×30μm, scale bar = 5μm) are shown. (B) For quantification of de novo protein synthesis in dendritic spines, labelled neurons were immunostained for homer to facilitate spine detection (not shown). The TAMRA intensity for each cell was measured in spines with detected co-localization of homer and GFP-SHANK3. Thereby homer-positive spines without a GFP-SHANK3 signal were used for normalization. The relative protein translation is plotted as ratio between the average TAMRA intensity of all GFP-SHANK3 positive and negative spines for each cell. Data is additionally normalized to the untreated control group. The quantification shows a significant reduction in relative protein translation after neuronal silencing but no significant effect of the SHANK3 variant (two-way ANOVA with Tukey post-hoc test, p = 0.012 for treatment, p = 0.529 for genotype, P = 95%). The data was obtained from three independent cultures (Ctrl: N(WT) = 14 cells, N(R12C) = 13 cells, N(L68P) = 13 cells; treatment: N(WT) = 16 cells, N(R12C) = 12 cells, N(L68P) = 11 cells).

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Cell Culture, Synthesized, Modification, Activity Assay, Labeling, Control, Variant Assay

Journal: bioRxiv

Article Title: Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

doi: 10.1101/2020.12.31.424970

Figure Lengend Snippet: CD spectra have been recorded between 175-260nm for a ~0.1 mg/mL solution of purified SHANK3 (1-676) variants in 10mM KH 2 PO 4 , 100mM KF, 0.5mM DTT, pH = 6.5 using a rectangular quartz cuvette with 1mm pathlength. For the CD spectra presented in , the dynode voltage is shown here as a function of wavelength.

Article Snippet: To test the conformational stability and accessibility of individual SHANK3 (1-676) variants to the protease trypsin, limited proteolysis was performed by incubating proteins in a molar ratio of 200:1 with Trypsin-EDTA (Gibco) at room temperature.

Techniques: Circular Dichroism, Purification

Journal: Electrophoresis

Article Title: A comparative study of CE-SDS, SDS-PAGE, and Simple Western: Influences of sample preparation on molecular weight determination of proteins.

doi: 10.1002/elps.202000199

Figure Lengend Snippet: Figure 1. Linear regression plots based on the logarithm of the MW versus the relative migration distance (Rf) respectively the reciprocal relative migration time (RMT) of MW marker proteins (Precision Plus ProteinTM Standard from Bio-Rad). (A) Linear regression of the data points while all points included on SDS-PAGE; (B) Linear regression of the data points, deviating points excluded on SDS-PAGE; (C) Residual plot of the data points out of (A); (D) Residual plot of the data points out of (B); (E) Linear regression of the data points while all points included on CE-SDS; (F) Linear regression of the data points, deviating points excluded on CE-SDS

Article Snippet: Molecular Weight Markers Maurice CE-SDS (ProteinSimple, a Bio-Techne Brand, San Jose, CA, USA, specified size range: 10–270 kDa) and ProteomeLabTM MW Sizing Standard (AB Sciex Germany GmbH, Darmstadt, Germany, specified size range: 10–225 kDa) were chosen as typical CE-SDS markers and Precision Plus ProteinTM Standard unstained (Bio-Rad Laboratories GmbH, Feldkirchen, Germany, specified size range: 10–250 kDa), Unstained Protein Standard (New England Biolabs GmbH, Frankfurt am Main, Germany, specified size range: 10–200 kDa) and BenchmarkTM Unstained Protein Ladder (Novex by life technologies, Thermo Fisher Scientific, MA, USA, specified size range: 10– 220 kDa) were chosen as typical SDS-PAGE markers.

Techniques: Migration, Marker, SDS Page

Journal: Electrophoresis

Article Title: A comparative study of CE-SDS, SDS-PAGE, and Simple Western: Influences of sample preparation on molecular weight determination of proteins.

doi: 10.1002/elps.202000199

Figure Lengend Snippet: Figure 2. Comparison of MW markers of five different man- ufacturers by 10% SDS-PAGE. The relevant lanes of the gel were reconstructed for better illustration using ChemDraw 19.0.1.28 (PerkinElmer Infor- matics, Inc.); a photo of the gel is shown in Figure S2 in the Supporting Information. Bio-Rad = Precision Plus ProteinTM Standard unstained by Bio-Rad; ProteinSim- ple = Molecular Weight Marker Maurice CE-SDS by ProteinSimple; Sciex = ProteomeLabTM MW Sizing Standard by Sciex; Biolabs = Unstained Protein Standard by New England Biolabs; Benchmark = BenchmarkTM

Article Snippet: Molecular Weight Markers Maurice CE-SDS (ProteinSimple, a Bio-Techne Brand, San Jose, CA, USA, specified size range: 10–270 kDa) and ProteomeLabTM MW Sizing Standard (AB Sciex Germany GmbH, Darmstadt, Germany, specified size range: 10–225 kDa) were chosen as typical CE-SDS markers and Precision Plus ProteinTM Standard unstained (Bio-Rad Laboratories GmbH, Feldkirchen, Germany, specified size range: 10–250 kDa), Unstained Protein Standard (New England Biolabs GmbH, Frankfurt am Main, Germany, specified size range: 10–200 kDa) and BenchmarkTM Unstained Protein Ladder (Novex by life technologies, Thermo Fisher Scientific, MA, USA, specified size range: 10– 220 kDa) were chosen as typical SDS-PAGE markers.

Techniques: Comparison, SDS Page, Molecular Weight, Marker