Protein fractions were analyzed by SDS-PAGE followed by either Coomassie blue staining or Western blotting. Proteins were separated on 3 to 8% tris-acetate NuPAGE mini gels (Thermo Fisher Scientific) and stained by SimplyBlue SafeStain solutions (Invitrogen) for 60 min. Gels were washed with deionized water for 60 min and then 3% NaCl for an additional 2 hours—overnight to achieve maximum sensitivity. Western blots were performed as described previously (16 (link)). The following primary antibodies were used in our analysis: BRCA1-C20 (SCBT, sc-642; α-BRCT), BRCA1-Ab1 (Calbiochem, OP92; α-RING), BRCA1-A8X9F (Cell Signaling Technology, #14823; α-RING), BARD1 (SCBT, sc-11438), ubiquitin-pAb (Enzo Life Sciences, ADI-SPA-200), RAD51 (SCBT, sc-8349), and β-actin (Sigma-Aldrich, A5441).
Protocol detail
Protein Analysis by SDS-PAGE and Western Blot
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Acetate
Actin
Antibodies
Brca1
Coomassie blue
Gels
Nacl
Proteins
Sds page
Sensitivity
Tris
Ubiquitin
Western blots
Corresponding Organization : Carilion Clinic
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Variable analysis
independent variables
- Protein fractions were analyzed by either Coomassie blue staining or Western blotting
- Protein separation and detection
- Proteins were separated on 3 to 8% tris-acetate NuPAGE mini gels (Thermo Fisher Scientific)
- Gels were stained by SimplyBlue SafeStain solutions (Invitrogen) for 60 min
- Gels were washed with deionized water for 60 min and then 3% NaCl for an additional 2 hours—overnight to achieve maximum sensitivity
- Western blots were performed as described previously (
16 (link)) - The following primary antibodies were used in our analysis: BRCA1-C20 (SCBT, sc-642; α-BRCT), BRCA1-Ab1 (Calbiochem, OP92; α-RING), BRCA1-A8X9F (Cell Signaling Technology, #14823; α-RING), BARD1 (SCBT, sc-11438), ubiquitin-pAb (Enzo Life Sciences, ADI-SPA-200), RAD51 (SCBT, sc-8349), and β-actin (Sigma-Aldrich, A5441)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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