Western blots were performed using the following antibodies: α FANCD2 (Santa Cruz Biotechnology; FI17), α Ku70 (Santa Cruz Biotechnology; A9), α PCNA (Santa Cruz Biotechnology; PC10), α phospho-(S1981)-ATM (Millipore), α ATM (GeneTex 2C1), α phospho-(S345)-Chk1 (Cell Signalling), α Chk1 (Santa Cruz Biotechnology, G4), α p21 (Santa Cruz Biotechnology, C19), α p53 (DO-1 and 1801) and α γH2AX (Upstate). α phospho (S824) KAP1 (Bethyl Laboratories), α KAP1 (Bethyl Laboratories), α Pol η (Santa Cruz Biotechnology; H-300). Incubation with secondary antibodies (Sigma) and ECL detection (Amersham GE Healthcare) were performed according to the manufacturers' instructions. Western blot images were taken with Image QuantLAS4000 (GE Healthcare), which allows capture and quantification of images within a linear range. These images were then quantified with the ImageJ software.
α chk1
Manufactured by Santa Cruz Biotechnology
α Chk1 is a lab equipment product manufactured by Santa Cruz Biotechnology. It is a key component involved in the regulation of cell cycle checkpoints.
Automatically generated - may contain errors
Lab products found in correlation
α kap1, by Fortis Life Sciences (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Ecl detection, by GE Healthcare (1 mentions)
α pcna, by Santa Cruz Biotechnology (1 mentions)
α actin, by Merck Group (1 mentions)
α γh2ax, by Merck Group (1 mentions)
α cdc45, by Santa Cruz Biotechnology (1 mentions)
T6557, by Merck Group (1 mentions)
α 4e bp1, by Cell Signaling Technology (1 mentions)
αγ tubulin, by Merck Group (1 mentions)
3 protocols using α chk1
1
Comprehensive Western Blot Analysis of DNA Damage Response
2
Chromatin Fractionation and Western Blot Analysis
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Cells were lysed and harvested with Laemmli buffer, followed by 8 min of incubation at 99°C. The chromatin fraction was obtained after a 5-min extraction with ice-cold CSK buffer [10 mM Pipes (pH 7.5), 100 mM NaCl, 300 mM sucrose, 1 mM EGTA, 3 mM MgCl2, and 2% Triton X-100]. The following antibodies were used: α-Chk1 at 1:1000 (Santa Cruz Biotechnology, sc-8408), α-γH2AX at 1:4000 (Millipore, 05-636), α-phospho-KAP1 Ser824 at 1:4000 (Bethyl Laboratories, A300-767A), α-Mus81 at 1:1000 (Santa Cruz Biotechnology, sc-53382), α-CDC45 at 1:1000 (Santa Cruz Biotechnology, sc-20685), α-KAP1 at 1:4000 (Bethyl Laboratories, A300-274A), α-phospho-RPA Ser4/8 at 1:8000 (Bethyl Laboratories, A300-245A), α-H2B (histone 2B) at 1:2000 (Santa Cruz Biotechnology, sc-515808), and α-actin at 1:20,000 (Sigma-Aldrich, A2066). Incubations with secondary antibodies (Jackson ImmunoResearch) and enhanced chemiluminescence (ECL) detection (GE Healthcare) were performed according to the manufacturers’ instructions. Western blot images were acquired with ImageQuant LAS4000 (GE Healthcare), which allows the capture and the quantification of images within a linear range.
3
Immunoblotting of Mammalian and Fission Yeast Proteins
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Samples for immunoblots of mammalian proteins were prepared by 3 × wash with cold 1 × PBS and kept at −80 °C until 2 × Laemmli buffer was added to make whole cell lysates. Total cell extracts for immunoblots of S. pombe proteins were made by TCA protein extraction53 (link). Antibodies for immunoblots were α-γtubulin (1:10000, T6557, Sigma-Aldrich), α-CHK1-P345 (1:1000, 2348, Cell Signaling Technology), α-CHK1 (1:200, DCS310.1, Santa Cruz), α−4EBP1 (1:2000, Cell Signaling Technology) α-phosphoAkt substrates (1:1000, 23C8D2, Cell Signaling Technologies), α-PSTAIRE, recognizing a motif in cdc2 (1:2000, Santa Cruz Biotechnology sc-53) and anti-peroxidase PAP1, against the TAP-tap (1:1000, Sigma P1291). Appropriate ECL and ECF kits were used for detection.
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