Initially, the cell protein lysates were separated on 10% or 12% SDS polyacrylamide gels, electrophoretically transferred to PVDF membranes (0.22 μm pore size; Millipore, USA). The transferred PVDF membrane was then incubated with rabbit anti‐p16 polyclonal antibody (10883‐1‐AP, Protein Tech, China), mouse anti‐CCNA2 monoclonal antibody (ab38, Abcam, USA), anti‐p53 polyclonal antibody (10442‐1‐AP, Protein Tech, China), or anti‐p21 polyclonal antibody (sc‐471, Santa Cruz Biotechnology, USA), followed by the respective secondary antibodies conjugated with horseradish peroxidase (HR) and subjected to a commercial enhanced chemiluminescence (ECL) kit (Pierce, USA). Protein loading was estimated using mouse anti‐tubulin monoclonal antibody (T5168, Sigma‐Aldrich, USA) or mouse anti‐GAPDH monoclonal antibody (60004‐1‐Ig, Protein Tech, China).
Sc 471
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-471 is a laboratory product offered by Santa Cruz Biotechnology. It is a piece of equipment designed for use in scientific research and experimentation. The core function of Sc-471 is to facilitate the performance of various laboratory procedures and analyses, but no further details about its intended use or capabilities can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Ab70250, by Abcam (1 mentions)
Ab31333, by Abcam (1 mentions)
Trans blot turbo transfer pack, by Bio-Rad (1 mentions)
Sc 23921, by Santa Cruz Biotechnology (1 mentions)
Image 6 quant las 500, by GE Healthcare (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Signalstain boost ihc detection reagent, by Cell Signaling Technology (1 mentions)
Ab32384, by Abcam (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
Anti mouse igg, by Beyotime (1 mentions)
T5168, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
4 protocols using sc 471
1
Western Blot Analysis of Cell Proteins
2
Western Blot Analysis of DNA Damage Responses
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Appropriate amounts of proteins (30 μg or 60 μg), extracted from a pool of 3 P1-cerebella and MEFs were loaded and separated by SDS-PAGE. Proteins were electro transferred to PVDF or nitrocellulose membranes (Trans-Blot Turbo Transfer Pack, BIO-RAD Laboratories, Hercules, CA) at the Trans-Blot Turbo Transfer System (BIO-RAD Laboratories). After blocking, membranes were incubated over night at +4°C with primary antibodies against DNA-PKcs and p53 (ab70250 and ab31333, Abcam, Cambridge, UK), ATM, p21 (sc-23921 and sc-471, Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53(Ser18) (9284, Cell Signaling Technology, Inc., Danvers, MA), β-actin and HSP70 (A5316 and H5147, Sigma-Aldrich, St Louis, MO). Membranes were probed for 1h at RT with appropriated HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). Immunoreactive bands were visualized using Amersham ECL Prime WB detection reagent (GE Healthcare Europe, Milan, Italy). Images were acquired using a Image 6 quant LAS 500 (GE Healthcare Europe), and densitometric analysis was performed using ImageJ software.
3
Immunohistochemical Analysis of Bcl-xL and p21
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The sections of mouse pancreas were processed further for immunohistochemistry (IHC). The sections were immersed in 1× Target Retrieval Solution (pH 6.0) (Dako, Glostrup, Denmark) for Bcl-xL and p21 staining. The samples were heated to 120°C in a decloaking chamber for antigen retrieval. The endogenous peroxidase activity was quenched by incubating with 3% hydrogen peroxide in methanol for 30 minutes. After sections were blocked in 5% goat serum for 1 hour, they were incubated with antibodies against Bcl-xL (1:200, #2764; Cell Signaling Technology, Danvers, MA) and p21 (1:400, sc-471; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. After rinsing with phosphate-buffered saline, the slides were incubated with secondary antibody (SignalStain Boost IHC detection reagent; Cell Signaling Technology) for 30 minutes. After washes with phosphate-buffered saline, the sections were incubated with 3,3’-diaminobenzidine for 30 seconds and counterstained with hematoxylin. In every staining set, negative controls were applied by omission of the primary antibody.
4
Western Blot Analysis of Protein Expression
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Total protein was extracted 72 h after the transfection using RIPA Lysis Buffer (Beyotime Institute of Biotechnology,). The proteins were separated by SDS-polyacrylamide gel (12%) and were transferred to polyvinylidenedifluoride membranes (0.2 µm pore size) (EMD Millipore, Bellerica, MA, USA) and were then detected with a rabbit anti-p21 polyclonal antibody (sc-471, 1:600; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), a rabbit anti-Cdk1 monoclonal antibody (ab32384, 1:1,000; Abcam, Cambridge, MA, USA) (used to detect dephospho-Cdk1 (Tyr15) which refers to active Cdk1 signalling pathways) (21 (link),22 (link)), a mouse anti-Gas2 monoclonal antibody (M01, 1:1,000; Abnova, Taipei, Taiwan) and a mouse anti-α-tubulin monoclonal antibody (T5168, 1:5,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG or anti-mouse IgG; Beyotime Institute of Biotechnology,) were diluted 3,000-fold, and the signals were detected by an enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.).
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