Whole cell lysates were prepared in RIPA buffer. Proteins were separated by SDS-PAGE under reducing conditions. BRCA1 protein was detected using mouse monoclonal anti-BRCA1 (SC-6954, 1:100, Santa Cruz Biotechnology). Protein bands were visualized using ECL substrates (BioRad, #170-5061) and imaged on Kodak X-OMAT 2000A.
Sc 6954
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-6954 is a research-grade laboratory equipment designed for scientific applications. It serves as a [core function], enabling researchers to [core function] in their investigations. The product specifications and technical details are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Sc 8349, by Santa Cruz Biotechnology (3 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
A21441, by Thermo Fisher Scientific (2 mentions)
Nb100 304, by GeneTex (2 mentions)
A11032, by Thermo Fisher Scientific (2 mentions)
Brca1, by Santa Cruz Biotechnology (2 mentions)
Ab2175, by Abcam (2 mentions)
Ecl substrate, by Bio-Rad (1 mentions)
X omat 2000a, by Kodak (1 mentions)
Sc 22760, by Santa Cruz Biotechnology (1 mentions)
Sc 7392, by Santa Cruz Biotechnology (1 mentions)
Sc 55538, by Santa Cruz Biotechnology (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab15850, by Abcam (1 mentions)
Sc 5286, by Santa Cruz Biotechnology (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Kpna2, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
23 protocols using sc 6954
1
BRCA1 Protein Expression Analysis
2
Western Blot and Immunoprecipitation Antibodies
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The following antibodies were used for western blot analysis: anti-MDC1 rabbit polyclonal antibody (R2, manufactured in our lab) [1 (link)], anti-KPNA2 mouse monoclonal antibody (sc-55538, Santa cruz, Dallas, TX, USA), anti-HA mouse monoclonal antibody (sc-7392, Santa Cruz), anti-GFP mouse monoclonal antibody (sc-5286, Santa Cruz), anti-Histone3 rabbit polyclonal antibody (ab1791, Abcam, Cambridge, UK), and anti-α-Tubulin mouse monoclonal antibody (sc-5286, Santa Cruz). For immunoprecipitation assay, anti-HA rabbit polyclonal antibody (F-7, Santa Cruz), anti-GFP mouse monoclonal antibody (sc-5286, Santa Cruz), anti-MDC1 rabbit polyclonal antibody (R2, manufactured in our lab), and anti-KPNA2 mouse monoclonal antibody (sc-55538, Santa cruz) were used. The following antibodies were used for immunofluorescence staining: anti-MDC1 rabbit polyclonal antibody (R2), anti-HA mouse monoclonal antibody (sc-7392, Santa Cruz), anti-RNF8 goat polyclonal antibody (ab15850, Abcam), anti-53BP1 rabbit polyclonal antibody (sc-22760, Santa Cruz), anti-BRCA1 mouse monoclonal antibody (sc-6954, Santa cruz), anti-γH2AX mouse monoclonal antibody (05-636-1, Millipore, Burlington, MA, USA), and anti-RNF168 rabbit polyclonal antibody (ABE367, Millipore).
3
Western Blot Analysis of Cellular Proteins
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Total proteins from cell lines were extracted in TNN buffer (50 mM Tris-Cl, pH 7.4; 1% NP-40; 150 mM NaCl; and 1 mM ethylenediaminetetraacetic acid [EDTA]) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 mM Na3VO4) and quantified using the Bradford method. Protein samples (15 μg) were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking non-specific antibody binding sites, the membrane was incubated overnight at 4 °C with monoclonal antibody against pBRCA1 (9009S, Cell Signaling, Danvers, MA, USA), BRCA1 (sc-6954, Santa Cruz Biotechnology, Paso Robles, CA, USA), cleaved caspase-3 (9664, Cell Signaling), KPNA2 (sc-55537, Santa Cruz Biotechnology), PARP (sc-7150, Santa Cruz Biotechnology), pAMPK (2535S, Cell Signaling), β-actin (A5316, Sigma-Aldrich, St. Louis, MO, USA), and lamin A/C (ab108922, Abcam, Cambridge, MA, USA). All the antibodies were used at a dilution of 1:1000. After incubation with peroxidase-conjugated secondary antibodies at 37 °C for 1 h, the protein bands were visualized using enhanced chemiluminescence reagent (GE Healthcare Biosciences, Piscataway, NJ, USA) and detected using the Amersham Imager 680 (GE Healthcare Biosciences). β-actin and lamin A/C were used for normalization.
4
Antibody Validation Protocol for Western Blot and IHC
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Primary antibodies used in this study were as follows: mouse anti-USP4 (66822; Proteintech1:1000 dilution for WB, 1:300 dilution for IHC); mouse anti-BRCA1 (sc-6954; Santa Cruz; 1:300 dilution for WB); rabbit anti-BRCA1(ab16780; Abcam;1:400 dilution for IHC); rabbit anti-BRCA1 (ab191042; Abcam;1:1000 dilution for WB); rabbit anti-BARD1 (NB100; Novus Biologicals; 1:1000 dilution for WB); mouse anti-BARD1 (sc-74559; Santa Cruz; 1:500 dilution for WB); mouse anti-Flag (F3165; Sigma; 1:5000 dilution for WB); rabbit anti-HA (3924 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-Myc (M4439; Sigma;1:5000 dilution for WB); mouse anti-ubiquitin (3936 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-α-His (2366 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-α-Tubulin (3873 S; Cell Signaling Technology; 1:3000 dilution for WB), rabbit anti-β-actin (AC026; ABclonal; 1:1000 dilution for WB); rabbit anti-GAPDH(5174 S; Cell Signaling Technology; 1:5000 dilution for WB). For WB, western blots were detected and analyzed using ChemiDoc Imaging system (Bio-Rad). The original WB blots were included in the supplementary information.
5
Western Blot Analysis of BRCA1 and PARP
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Western blot analysis was performed as described previously [23 (link)]. Briefly, cell extracts were prepared using Laemmli buffer. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The following antibodies were used for immunoblotting: anti-β-actin (dilution 1:10000; A2228, Sigma-Aldrich), anti-BRCA1 (dilution 1:300; MAB22101, R&D systems (Minneapolis, MN, USA)), anti-BRCA1 (D-9)(dilution 1:100; sc-6954, Santa Cruz Biotechnology (Dallas, TX, USA)), and anti-PARP (dilution 1:2000; #9542, Cell Signaling Technology (Danvers, MA, USA)). Immune complexes were detected using a horseradish peroxidase-linked secondary antibody (Cytiva, Tokyo, Japan), and immunoreactive proteins were visualized using a chemiluminescence kit (Merck, Branchburg, NJ, USA) and the ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA). Image quantification was performed using ImageJ software (NIH, Rockville, MD, USA).
6
Immunoblotting Analysis of Cellular Proteins
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Two days after siRNA transfection, cells were harvested and lysed in 50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM NaF, 30 mM sodium pyrophosphate, 1 mM Na3VO4, 10 mM 2-glycerophosphate, 10 μg/mL leupeptin, 5 μg/mL aprotinin, 5 μg/mL pepstatin, and 20 mM microcystin-LR. Immunoblotting was done using the following primary antibodies: rabbit polyclonal ZC3H18 (1:1000, A304-682A, Bethyl Laboratories Inc.); mouse monoclonal BRCA1 (1:2000, sc-6954, Santa Cruz Biotechnology); mouse monoclonal E2F1 (1:500, ab4070, Abcam); rabbit polyclonal E2F4 (1:1000, NBP1-21374, Novus Biologicals); mouse monoclonal DNMT1 (1:5000, ab13537, Abcam); mouse monoclonal HSP90 (D. Toft, Mayo Clinic, H9010), and rabbit monoclonal HA-tag (1:1000, CST-3724S, Cell Signaling Technology). Secondary antibodies used were: horseradish peroxidase-conjugated anti-mouse immunoglobulin G (1:2000 for BRCA1 and 1:10,000 for all other primary antibodies, 7076 S, Cell Signaling Technology) and anti-rabbit immunoglobulin G (1:16,000 for ZC3H18 and 1:10,000 for all other primary antibodies, 7074 S, Cell Signaling Technology).
7
Immunostaining for DNA Damage Response
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Cells were grown on sterile 12 mm glass coverslips, fixed in 3% formaldehyde in PBS for 15 min at room temperature, washed once in PBS, permeabilized for 5 min at room temperature in PBS supplemented with 0.2% Triton X-100 (Sigma-Aldrich), and washed twice in PBS. All primary and secondary antibodies (Alexa fluorophores; Life Technologies) were diluted in filtered DMEM containing 10% FBS and 0.02% Sodium Azide. Antibody incubations were performed for 2 h at room temperature. After antibody incubations, coverslips were washed once with PBS and incubated for 10 min with PBS containing 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, 0.5 μg/ml) at room temperature to stain DNA. After three washing steps in PBS, coverslips were briefly washed with distilled water and mounted on 6 μl Mowiol-based mounting media. The following primary antibodies were used for immunostaining: H2AX Phospho S139 (mouse, 613401, 1:1,000; BioLegend), 53BP1 (mouse, Upstate MAB3802, 1:1,000), H4K20me2 (rabbit, ab9052, 1:100; Abcam), H4K20me1 (rabbit, ab9051, 1:200; Abcam), BRCA1 (mouse, sc-6954, 1:100; Santa Cruz), Cyclin A (mouse, sc-271682, 1:100; Santa Cruz), and RAD51 (rabbit, 70-002, 1:1,000; Bioacademia).
8
Antibody Characterization in Protein Complexes
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9
Western Blot Analysis of DNA Damage Signaling
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Cell lysates were prepared by sonicating in UTB buffer (8 M urea, 50 mM Tris HCl [pH 7.4], 150 mM β-mercaptoethanol). Following protein concentration determination using the Bradford reagent (Bio-Rad), lysates were suspended in 2× Laemmli sample buffer (Bio-Rad) and heated to 95°C for 10 min. Lysates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using standard procedures.
The following primary antibodies were used for Western blotting: anti-K8α/K-bZIP (SAB5300152; Sigma-Aldrich), anti-ORF6/SSB (provided by Gary Hayward), anti-K8.1A (in-house), anti-β-actin (A2228; Sigma-Aldrich), anti-ATM (2873; Cell Signaling), anti-phospho-ATM (S1981) (AF1655; R&D Systems), anti-CHK2 (2662; Cell Signaling), anti-phospho-CHK2 (T68) (2661; Cell Signaling), anti-RPA32 (NA19L; Calbiochem), anti-phospho-RPA32 (S4/S8) (A300-245A; Bethyl), anti-H2AX (7631; Cell Signaling), anti-γH2AX (S139) (05-636; Merck Millipore), anti-MRE11 (GTX70212; Genetex), anti-NBS1 (GTX70222; Genetex), anti-RAD50 and anti-KU80 (sc-6954; Santa Cruz), and anti-KU70 (ab83501; Abcam). Goat anti-mouse and swine anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako Laboratories) and ECL detection reagent (GE Healthcare) were used to visualize proteins on a Fusion SL chemiluminescence imaging system (Vilber Lourmat).
The following primary antibodies were used for Western blotting: anti-K8α/K-bZIP (SAB5300152; Sigma-Aldrich), anti-ORF6/SSB (provided by Gary Hayward), anti-K8.1A (in-house), anti-β-actin (A2228; Sigma-Aldrich), anti-ATM (2873; Cell Signaling), anti-phospho-ATM (S1981) (AF1655; R&D Systems), anti-CHK2 (2662; Cell Signaling), anti-phospho-CHK2 (T68) (2661; Cell Signaling), anti-RPA32 (NA19L; Calbiochem), anti-phospho-RPA32 (S4/S8) (A300-245A; Bethyl), anti-H2AX (7631; Cell Signaling), anti-γH2AX (S139) (05-636; Merck Millipore), anti-MRE11 (GTX70212; Genetex), anti-NBS1 (GTX70222; Genetex), anti-RAD50 and anti-KU80 (sc-6954; Santa Cruz), and anti-KU70 (ab83501; Abcam). Goat anti-mouse and swine anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako Laboratories) and ECL detection reagent (GE Healthcare) were used to visualize proteins on a Fusion SL chemiluminescence imaging system (Vilber Lourmat).
10
Immunoprecipitation and Immunoblotting Protocol
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Immunoprecipitation and immunoblotting was carried out as described previously (40 (link)). Briefly, cells were harvested and lysed in NTEN buffer (0.5% NP40, 50 mM Tris–HCl pH 8.0, 2 mM EDTA, 150 mM NaCl and 10% glycerol) supplemented with a complete protease inhibitor cocktail (Roche). For the immunoprecipitation assays, 1mg protein from the cell lysates was incubated with anti-HA (Roche) antibody and separated using protein G beads. The proteins were resolved on a 6% SDS-PAGE gel and transferred onto PVDF membrane (Millipore). The membranes were blocked using 5% skimmed milk in TBST buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, and 0.1% Tween-20) and incubated with primary antibody for 12 h at 4°C. The primary antibodies against FANCM (1:50, sc-101389), BRCA-1 (1:100, sc-6954), MCM3 (1: 500, sc-365616) that were used for western blot analysis were purchased from Santa Cruz. Anti-MRE11 (1:2000, 611366) antibodies were obtained from BD Biosciences; and the rabbit polyclonal antibody for FANCJ (1:1000, ab49657) was obtained from Abcam. Anti-HA antibody (1:2000, 12CA5) was obtained from Roche. The membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, developed by chemiluminescence, and imaged using Chemidoc (GE healthcare LAS 4000).
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