Journal: RSC Advances

Article Title: N- and C-terminal regions of the small heat shock protein IbpA from Acholeplasma laidlawii competitively govern its oligomerization pattern and chaperone-like activity

doi: 10.1039/c9ra10172a

Figure Lengend Snippet: Fig. 4 Interaction of truncated AlIbpA proteins with insulin in vitro assessed with pull down assay. Proteins were mixed in PBS in weights ratio of 1 : 1 and after 30 min incubation at 37 C were purified on Ni- NTA agarose and elution fractions were separated with 17.5% SDS- PAGE.

Article Snippet: Four hours aer induction, cells were harvested by centrifugation and stored at 20 C. All proteins with N-terminal His6-tag peptide were puried using affinity chromatography on Ni-NTA sepharose gravity-ow columns (Qiagen) following the protocol described previously.45 E. coli cell free extracts were prepared from the frozen cells (see above) by resuspending the cells in disruption buffer DB [50 mM Tris–HCl pH 7.4, 50 mM KCl, 500 mM NaCl, 5 mMMgCl2, 1 mM EDTA, 2 mM benzamidine, 1 mM phenylmethylsulfonyl uoride (PMSF)] and broken by sonication.

Techniques: In Vitro, Pull Down Assay, Incubation, SDS Page

Journal: Methods in Molecular Biology

Article Title: P-Type ATPases

doi: 10.1007/978-1-4939-3179-8

Figure Lengend Snippet: Fig. 1 The principle of calmodulin-affi nity chromatography. ( a ) Detergent - solubilized plasma membrane proteins are applied to a column packed with calmodulin coupled to Sepharose 4B, in the presence of 0.1 mM CaCl 2 . Calmodulin interacts reversibly with the PMCA molecules in the presence of Ca 2+ . ( b ) Proteins binding calmodulin with high affi nity, such as the PMCA, will be retained on the column, as they interact with the immobilized calmodulin. Non-binding proteins are eluted by a washing of the column with a Ca 2+ -containing buffer. ( c ) Proteins binding specifi cally and reversibly to calmodulin, such as the PMCA, can now be eluted from the column by exchanging the Ca 2+ -containing buffer for a Ca 2+ -free buffer containing EDTA

Article Snippet: Two 10 cm × 0.5 cm (inner diameter) Glass Econo-Column ® columns for gravity fl ow chromatography (Bio-Rad, Hercules, CA, USA) with outlet tubing of 1 and 2 mL dead-volume, respectively.

Techniques: Chromatography, Clinical Proteomics, Membrane, Binding Assay

Journal: Methods in Molecular Biology

Article Title: P-Type ATPases

doi: 10.1007/978-1-4939-3179-8

Figure Lengend Snippet: Fig. 1 Relative concentration of fatty acid species before and after LEC of a SERCA preparation. The relative concentrations of fatty acid species determined by gas chromatography of methylated fatty acids from extracted samples show a strong enrichment to ~85 % target lipid after LEC. The following samples were analyzed: DOC SR, deoxycholate-extracted sarcoplasmic reticulum (SR) micro- somes before solubilization; Solub. SR, C 12 E 8 solubilized SR microsomes enriched in SERCA; DOPC exchange, solubilized SERCA after LEC with DOPC as target lipid; DOPC strip, DOPC exchanged SERCA after sucrose step-gradient spin to strip the sample from bulk lipids. Error bars correspond to the standard deviation of results from three independent experiments. Adapted fi gure from [ 8 ]

Article Snippet: Two 10 cm × 0.5 cm (inner diameter) Glass Econo-Column ® columns for gravity fl ow chromatography (Bio-Rad, Hercules, CA, USA) with outlet tubing of 1 and 2 mL dead-volume, respectively.

Techniques: Concentration Assay, Gas Chromatography, Methylation, Stripping Membranes, Standard Deviation

Journal: Methods in Molecular Biology

Article Title: P-Type ATPases

doi: 10.1007/978-1-4939-3179-8

Figure Lengend Snippet: Fig. 1 Chromatograms from size exclusion chromatography of the nanodisc preparation. (a) Chromatograms of first run. (b) First run, zoom. (c) Second run of the fraction corresponding to one NKA molecule per nanodisc. (d) Second run of the fraction corresponding to two NKA molecules per nanodisc

Article Snippet: Two 10 cm × 0.5 cm (inner diameter) Glass Econo-Column ® columns for gravity fl ow chromatography (Bio-Rad, Hercules, CA, USA) with outlet tubing of 1 and 2 mL dead-volume, respectively.

Techniques: Size-exclusion Chromatography