Journal: Journal of proteome research

Article Title: Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies

doi: 10.1021/acs.jproteome.0c00772

Figure Lengend Snippet: Product Specifications for Various Streptavidin Bead Products Used in Proteomics Workflows a

Article Snippet: Vector Laboratories , MagnaLINK streptavidin magnetic beads , SPM , M-1003–010 , 2.8 , 60% magnetite content , free biotin: ≥10 nmol/mg biotinylated IgG: ≥0.8 nmol/mg biotinylated oligonucleotides: ≥0.75 nmol/mg , fluorescein-biotin assay.

Techniques: Binding Assay, Magnetic Beads, Plasmid Preparation

Journal: Journal of proteome research

Article Title: Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies

doi: 10.1021/acs.jproteome.0c00772

Figure Lengend Snippet: Categorization of Streptavidin Bead Products Used in This Study Based on Their Behavior in Binding Buffer

Article Snippet: Vector Laboratories , MagnaLINK streptavidin magnetic beads , SPM , M-1003–010 , 2.8 , 60% magnetite content , free biotin: ≥10 nmol/mg biotinylated IgG: ≥0.8 nmol/mg biotinylated oligonucleotides: ≥0.75 nmol/mg , fluorescein-biotin assay.

Techniques: Binding Assay, Magnetic Beads

Journal: Journal of proteome research

Article Title: Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies

doi: 10.1021/acs.jproteome.0c00772

Figure Lengend Snippet: General considerations for evaluating the fit-for-purpose of streptavidin bead products and reporting key parameters to promote repeatability among studies. Key features to consider when implementing streptavidin beads into a sample preparation workflow are shown and include physical bead characteristics, bead handling characteristics and performance, and whether manual or automated sample processing is required. Technical details that should be included when reporting data from studies that implement streptavidin-bead-based enrichment are indicated with a black star.

Article Snippet: Vector Laboratories , MagnaLINK streptavidin magnetic beads , SPM , M-1003–010 , 2.8 , 60% magnetite content , free biotin: ≥10 nmol/mg biotinylated IgG: ≥0.8 nmol/mg biotinylated oligonucleotides: ≥0.75 nmol/mg , fluorescein-biotin assay.

Techniques: Sample Prep

Journal: Journal of proteome research

Article Title: Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies

doi: 10.1021/acs.jproteome.0c00772

Figure Lengend Snippet: Intralot variation in binding capacity of streptavidin bead results in differences in number of proteins identified in a CSC experiment. (A) Total number of proteins and cell surface N-glycoproteins identified containing consensus motif (NxS/T/V/c, where x ≠ P) in CSC experiments (1000 μg total peptide) when using 100 μL each of two different lots of GenScript streptavidin MagBeads (C44251904 and C44241809). (B) Binding capacity as reported by GenScript. The orange dashed line highlights the minimum binding capacity necessary to pass quality control (60 nmol/mL). (C) Peak area replicate comparison view for precursors M, M+1, and M+2 for streptavidin peptides YDSAPATDGSGTALGWTVAWK, NAHSATTWSGQYVGGAEAR, INTGWLLTSGTTEANAWK, and STLVGHFTFTK (technical replicates = 4) detected in the CSC results from panel A.

Article Snippet: Vector Laboratories , MagnaLINK streptavidin magnetic beads , SPM , M-1003–010 , 2.8 , 60% magnetite content , free biotin: ≥10 nmol/mg biotinylated IgG: ≥0.8 nmol/mg biotinylated oligonucleotides: ≥0.75 nmol/mg , fluorescein-biotin assay.

Techniques: Binding Assay

Journal: Journal of proteome research

Article Title: Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies

doi: 10.1021/acs.jproteome.0c00772

Figure Lengend Snippet: AVIDITY assay is compatible with a range of streptavidin bead products. (A) HABA titration curves for GenScript streptavidin MagBeads (lot C44261906), Cytiva Sera-Mag Magnetic neutravidin-coated particles (lot 17015347), and New England Biolabs streptavidin magnetic beads (lot 10051761). (B) Absorbances of free HABA of five different bead types at 25 nmol of HABA are shown as means with SD error bars (n = 3). An absorbance range of 0.9 to 1.5 was suitable for all beads tested. (C) Biotin titration curves for two different lots of GenScript streptavidin MagBeads (lots C44261906 and C44242003) and one lot of Cytiva Sera-Mag SpeedBeads neutravidin-coated magnetic beads (lot 17015347). Changes in absorbance (Δ absorbance) as determined by subtracting the control absorbance reading (beads incubated with HABA) from the experimental absorbance reading (beads incubated with HABA and d-biotin) are shown as means with SD error bars (n = 3).

Article Snippet: Vector Laboratories , MagnaLINK streptavidin magnetic beads , SPM , M-1003–010 , 2.8 , 60% magnetite content , free biotin: ≥10 nmol/mg biotinylated IgG: ≥0.8 nmol/mg biotinylated oligonucleotides: ≥0.75 nmol/mg , fluorescein-biotin assay.

Techniques: Titration, Magnetic Beads, Incubation

Journal: Journal of proteome research

Article Title: Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies

doi: 10.1021/acs.jproteome.0c00772

Figure Lengend Snippet: Binding capacity of streptavidin beads as measured by the AVIDITY assay is predictive of proteomics results. (A) Total numbers of proteins and cell surface N-glycoproteins identified containing the consensus motif (NxS/T/V/c, where x ≠ P) in CSC experiments (1000 μg of total peptide) when using 100 μL each of two different lots of GenScript streptavidin MagBeads and one lot each for Cytiva Sera-Mag SpeedBeads neutravidin-coated magnetic beads (lot 17015347), Cytiva Sera-Mag streptavidin-coated magnetic beads (lot 17013370), and Invitrogen Dynabeads MyOne streptavidin C1 (lot 00945048) are shown. (B) Binding capacity as determined by the AVIDITY assay using 25 nmol HABA and 100 nmol d-biotin shown as means with SD error bars (n = 3).

Article Snippet: Vector Laboratories , MagnaLINK streptavidin magnetic beads , SPM , M-1003–010 , 2.8 , 60% magnetite content , free biotin: ≥10 nmol/mg biotinylated IgG: ≥0.8 nmol/mg biotinylated oligonucleotides: ≥0.75 nmol/mg , fluorescein-biotin assay.

Techniques: Binding Assay, Magnetic Beads

Journal: bioRxiv

Article Title: Cancer missense mutations in the BRC repeats of BRCA2 protein disrupt RAD51 binding and activity leading to chemotherapeutic sensitivity

doi: 10.1101/2021.09.24.461749

Figure Lengend Snippet: (A) Schematic of reaction to assay BRC repeat stimulation of RAD51-ssDNA complex formation by EMSA. RAD51 was pre-incubated with increasing amounts of BRC protein for 15 minutes then radiolabeled ssDNA (dT40) was added for 40 minutes. All reactions were incubated at 37 degrees and visualized on a 6% TAE polyacrylamide gel. (B) Autoradiograms of EMSA gels depicting increasing concentration of BRC proteins: BRC2, BRC2-S1221P, BRC7, BRC7-T1980I incubated with 10 nM RAD51, and 400 pM ssDNA (Oligo dT40*) Gel represents no protein (lane 1), RAD51 (lane 2), BRC2 or BRC7 (lane 3-6) and BRC2-S1221P or BRC7-T1980I (lane 7-10). (C) Quantification of BRC-RAD51-ssDNA complexes in the presence of increasing concentrations of BRC2, BRC2-S1221P, BRC7, BRC7-T1980I calculated from gels shown in B. Error bars represent the S.D. for two independent experiments. (D) Schematic of biotinylated DNA pull-down assay. Purified BRC proteins were pre-incubated with increasing concentrations of purified RAD51 for 10 min. Biotinylated ssDNA (167-mer) was then added for 10 minutes to allow nucleoprotein filament formation and captured on magnetic streptavidin beads. The beads were then washed, eluted in sample buffer, and analyzed by SDS-PAGE and western blotting (E) Western blot depicting amount of RAD51 pulled down and eluted from biotin-DNA-BRC-RAD51 complexes. RAD51 concentrations from 0-100 nM were pre-incubated with 80 nM of BRC peptide, incubated with biotin-ssDNA, washed extensively, and then eluted. Gel represents RAD51 alone (lane 1-3), RAD51+BRC2 (lane 4-6), RAD51+BRC2-S1221P (lane 7-9), RAD51+BRC7 (lane 10-12), RAD51+BRC7-T1980I (lane 13-15).

Article Snippet: The DNA-protein complexes were then captured by adding 2.5 µL of pre-washed MagnaLink Streptavidin magnetic beads (SoluLink) in buffer SW (Buffer S supplemented with 0.1% Igepal CA-630 and 0.5% Triton X-100).

Techniques: Incubation, Concentration Assay, Pull Down Assay, Purification, SDS Page, Western Blot