Cell extracts were prepared in RIPA lysis buffer (NEB) supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma, NEB). Immunoblots were performed using standard procedures. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4–12% gradient gels, Invitrogen) and subsequently transferred onto nitrocellulose membranes. Primary antibodies for MLH1 (554073, BD Pharmigen), MSH2 (ab52266, Abcam), MSH3 (ab69619, Abcam), Tubulin (3873, Cell Signalling) and ß-Actin (A5060, Sigma) were used at 1:1000. Secondary antibodies were used at 1:5000 (HRP-conjugated goat anti-mouse or anti-rabbit IgG from Jackson Immunochemicals). Immunoblots were imaged using a Curix 60 (AGFA) table-top processor. Uncropped immunoblots can be found in the Source Data file (for Fig. 3b ) and Supplementary Fig. 5 .
Ripa lysis buffer
Manufactured by New England Biolabs
RIPA lysis buffer is a commonly used detergent-based cell lysis and protein extraction solution. It contains a combination of ionic and non-ionic detergents that effectively lyse cells and solubilize proteins for subsequent analysis.
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Lab products found in correlation
Protease inhibitor, by Merck Group (8 mentions)
Phosphatase inhibitor, by Merck Group (7 mentions)
Curix 60, by AGFA HealthCare (5 mentions)
Sds page, by Thermo Fisher Scientific (3 mentions)
Gradient gel, by Thermo Fisher Scientific (3 mentions)
A5060, by Merck Group (2 mentions)
Protein assay dye reagent, by Bio-Rad (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Amersham protran nitrocellulose membranes, by Cytiva (2 mentions)
Fancd2 epr2302, by Abcam (2 mentions)
Ps824 kap1, by Fortis Life Sciences (2 mentions)
Hrp conjugated goat anti mouse rabbit igg, by Merck Group (2 mentions)
Asciz, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Ab52266, by Abcam (1 mentions)
Ab69619, by Abcam (1 mentions)
Exo1 a302 639a, by Fortis Life Sciences (1 mentions)
Atm 2c1, by Santa Cruz Biotechnology (1 mentions)
Chk1 dcs 310, by Santa Cruz Biotechnology (1 mentions)
Ps15 p53, by Cell Signaling Technology (1 mentions)
11 protocols using ripa lysis buffer
1
Immunoblotting with Lysis and Antibodies
2
Protein Extraction and Western Blotting Protocol
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Cells were lysed in RIPA lysis buffer (New England Biolabs), sonicated and protein concentrations were measured using the Protein Assay Dye Reagent (Biorad). Samples were mixed with NuPAGE LDS Sample Buffer (Invitrogen), boiled for 5 minutes at 98°C and proteins were separated on SDS-PAGE gels and transferred onto Amersham™ Protran nitrocellulose membranes (0.45μm, Cytiva) for confirmation of the knockout status of cell lines, or onto Amersham™ Hybond PVDF membrane (0.2 μm, Cytiva) for the identification of Pt-alkyne-53 interactors. After 1 hour of blocking in 5% milk in TBS-T (0.1% Tween 20 in 1x Tris-buffered saline), membranes were incubated with primary antibodies at 4°C overnight. Primary antibodies used were against FANCD2 (diluted 1:1,000, EPR2302 Abcam), XPF (diluted 1:500, 3F2/3 Santa Cruz), PCNA (diluted 1:500, PC10 Santa Cruz) and H3 (diluted 1:15000, ab1791 Abcam), and as a loading control, against Tubulin (diluted 1:10,000, DM1A Cell Signaling). Anti-mouse and anti-rabbit HRP-conjugated goat secondary antibodies (Jackson Immunochemicals) were used at a final dilution of 1:5,000. Immunoblots were imaged using a Curix 60 (AGFA) table-top processor.
3
Immunoblot Analysis of Cas9 Protein
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Cell extracts were prepared in RIPA lysis buffer (NEB) supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma, NEB). Immunoblots were performed using standard procedures. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (3–8% gradient gels, Invitrogen) and subsequently transferred onto nitrocellulose membranes. Primary antibodies for Cas9 (7A9-3A3, Cell Signaling Technology #14697) and ß-Actin (A5060, Sigma) were used at 1:1,000. Secondary antibodies were used at 1:5,000 (HRP-conjugated goat anti-mouse or anti-rabbit IgG from Jackson Immunochemicals). Immunoblots were imaged using a Curix 60 (AGFA) table-top processor.
4
Western Blotting Using Gradient SDS-PAGE
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Cell extracts were prepared in RIPA lysis buffer (NEB) supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma, NEB). Immunoblots were performed using standard procedures. Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (4–12% gradient gels; Invitrogen) and subsequently transferred onto nitrocellulose membranes. Details of primary antibodies used for western blotting (WB) and immunofluorescence (IF) are described in Supplementary Table 1 . Secondary antibodies were used at 1:5000 (HRP-conjugated goat anti-mouse, rabbit or goat IgG from Jackson Immunochemicals) for WB and 1:600 or 1:1000 for IF (AlexaFluorophores).
5
Immunoblotting Protocol for DNA Repair Proteins
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Cell extracts were prepared in RIPA lysis buffer (NEB) supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma, NEB). Immunoblots were performed using standard procedures. Protein samples were separated by SDS–PAGE (4–12% gradient gels; Invitrogen) and subsequently transferred onto nitrocellulose membranes. All primary antibodies were used at 1:1000 dilution with the exception of BLM (1:500) and RMI1 (1:5000). Secondary antibodies were used at 1:5000. The following antibodies were used: FANCC clone 8F3 (Merck Millipore), FANCD2 EPR2302 (Abcam), FANCI A301-254 (Bethyl laboratories), NQO1 clone A180 (Cell Signaling), RMI1 (Proteintech), BLM clone C-18 (Santa Cruz), MUS81 clone MTA30 2G10/3 (Abcam), p21 clone F-5 (Santa Cruz), β-Actin clone 20–33 (Sigma), Tubulin clone DM1A (Cell Signaling), HRP-conjugated goat anti-mouse, rabbit or goat IgG (Jackson Immunochemicals). The p53 antibody (PAB 421) was from Cancer Research UK. Uncropped immunoblot images are shown in Supplementary Fig. 7 .
6
Immunoblot Analysis of DNA Repair Proteins
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Cell extracts were prepared using RIPA lysis buffer (NEB) with protease (Sigma) and phosphatase (Sigma) inhibitors. Immunoblots were performed using standard procedures. Samples containing proteins were separated using SDS PAGE 4–12% gradient gels (Invitrogen) and transferred onto nitrocellulose membranes. The membranes were incubated with primary and secondary antibodies. The primary antibodies were NUDT1 (NB100-109, Novus Biologicals), CHK2 (05–649, Millipore), POLM (C1, Santa Cruz), EXO1 (A302-639A, Bethyl Laboratories), FANCC (MABC524- clone 8F3, Millipore), POLE (GTX132100, GeneTex), Actin (A5060, SIGMA), NEIL1 (12145-1-AP, Proteintech), POLB (ab26343, Abcam), and MSH6 (D60G2, Cell Signalling). Catalogue numbers and working dilutions for antibodies are provided in Supplementary Table 1 . Uncropped immunoblot images are shown in Supplementary Fig. 9 .
7
Immunoblotting of DNA Damage Response
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Cells were extracted in RIPA lysis buffer (NEB) supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma, NEB). Immunoblots were performed using standard procedures. Protein samples were separated by SDS–PAGE (3–8% gradient gels; Invitrogen), and subsequently transferred onto nitrocellulose membranes. All primary antibodies were used at 1:1000 dilution, except for P-S957-SMC1 that was used at 1:400, and secondary antibodies at 1:5000. The following antibodies were used: ATM 2C1 (Santa Cruz), P-S1981-ATM (10H11.E12; NEB), ASCIZ (Millipore), P-S824-KAP1 (Bethyl Laboratories, Inc), KAP1 (Bethyl Laboratories, Inc), P-S15-p53 (16G8; NEB), P-S957-SMC1 (5D11G5; Millipore), SMC1 (Abcam), P-S317-CHK1 (NEB), CHK1 (DCS-310; Santa Cruz), FANCD2 (EPR2302; Abcam), p95/NBS (NEB), β-actin (Sigma), 53BP1 (H300; Santa Cruz), CHK1 (DCS-310; Santa Cruz), HRP-conjugated goat anti-mouse/rabbit IgG (Sigma).
8
Western Blot Analysis of DNA Damage Response
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Cells were extracted in RIPA lysis buffer (NEB) supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma, NEB). Western blots were performed using standard procedures. Protein samples were separated by SDS–PAGE (3–8% or 4–12% gradient gels; Invitrogen), and subsequently transferred onto nitrocellulose membranes. All primary antibodies were used at 1:1000 dilution and secondary antibodies at 1:5000. The following antibodies were used: ATM (Santa Cruz), ASCIZ (Millipore); p95 (known as NBS1) (NEB), β-actin (Sigma), pS15-p53 (Cell Signalling), pS824-KAP1 (Bethyl Labs), total p53 (Pab-421; CR-UK generated antibody) and HRP-conjugated goat anti-mouse or rabbit IgG (Sigma).
9
Probing Signaling Cascades in NSCs
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For Western Blots (WB), NSCs were extracted in RIPA lysis buffer (NEB) supplemented with protease inhibitors (Sigma). For immunoprecipitation (IP) cells were lysed in IP buffer (20mM sodium phosphate buffer, 1 mM EDTA, 0.2% NP40, 150 mM NaCl supplemented with Na-orthovanadate, PMSF, NaF and protease inhibitor mixture [Sigma]). After sonication and centrifugation, supernatant was incubated overnight at 4°C with Pdgfra antibody, followed by 2-hr incubation with Dynabeads M-280 (Invitrogen), washed with IP buffer (0 mM NaCl, 150 mM NaCl and 1 M NaCl) and eluted in Laemmli sample buffer. All primary antibodies were used at 1:1000 dilution and secondary antibodies at 1:10000. The following antibodies were used: p53, Pdgfra, p-Pdgfra, p-Chk2, p-Akt, Akt (all Cell Signaling); Myc-9E10 (CRUK); Smc1 (Abcam); p-Smc1, Chk2, Tubulin (all Merck); p-Kap1, Kap1 (both Bethyl Labs); Atm (Santa Cruz), p-Atm (Epitomics), p-p53, Actin, GAPDH, HRP-conjugated goat anti-mouse/rabbit IgG (all Sigma).
10
Immunoblotting for DNA Repair Proteins
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Cell extracts were prepared in RIPA lysis buffer (NEB), supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma). Immunoblots were performed using standard procedures. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 3-8% or 4-12% gradient gels (Invitrogen) and subsequently transferred onto PVDF membranes. The following antibodies were used in 5% milk: BRCA1 (1:1000, Santa Cruz, #sc-6954), a-Tubulin (1:1000, CellSignaling, #3873), RPA32 (1:1000, abcam, #ab2175), Vinculin (1:5000, Sigma-Aldrich, #MAB3574). Secondary antibodies (HRP-conjugated goat anti-mouse or anti-rabbit IgG, Jackson Immunochemicals) were used at a 1:5000 dilution. Immunoblots were imaged using a Curix60 (AGFA) table-top processor.
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