Protein lysates were prepared, quantified, and equally loaded on 4%–20% Tris-HCL Criterion Gel (Bio-Rad). Proteins were transferred onto PVDF membrane (Bio-Rad), blocked, and stained overnight using the following primary antibodies: anti-CHD8 (1:1000, Bethyl, A301-2214A), HRP conjugated anti-GAPDH (1:5000, Cell Signaling Technology, 3683), and HRP conjugated anti-ACTB (1:1000, Cell Signaling Technologies, 5125). Membranes were washed and stained with secondary antibodies: HRP-conjugated secondary antibodies (1:10,000, Cell Signaling Technology, 7074 and 7076). Membranes were washed, developed using SuperSignal West Femto Substrate (Pierce), and imaged on a gel imager (Bio-Rad).
Hrp conjugated anti gapdh
Manufactured by Cell Signaling Technology
Sourced in United States
HRP conjugated anti-GAPDH is a primary antibody conjugated with horseradish peroxidase (HRP). It recognizes and binds to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Cell Signaling Technology (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Gel imager, by Bio-Rad (1 mentions)
Criterion tris hcl gel, by Bio-Rad (1 mentions)
Anti chd8, by Fortis Life Sciences (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Anti g6pd, by Cell Signaling Technology (1 mentions)
Anti atm, by GeneTex (1 mentions)
Ecl plus detection reagent, by GE Healthcare (1 mentions)
Hrp conjugated anti β actin, by Merck Group (1 mentions)
Curix 60 developer, by AGFA HealthCare (1 mentions)
Hrp conjugated goat anti rabbit igg, by GE Healthcare (1 mentions)
Hrp conjugated goat anti mouse igg, by GE Healthcare (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Anti sirt6, by Cell Signaling Technology (1 mentions)
Anti sirt3, by Cell Signaling Technology (1 mentions)
Anti acetylated lysine, by Cell Signaling Technology (1 mentions)
Anti sirt2, by Cell Signaling Technology (1 mentions)
Anti sirt5, by Cell Signaling Technology (1 mentions)
7 protocols using hrp conjugated anti gapdh
1
Western Blot Analysis of CHD8 Protein
2
Cellular Protein Expression Analysis by Western Blotting
3
Western Blot Analysis of Proteasome Subunits
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For Western blot analysis, 10–20 μg of protein were subjected to electrophoresis on 10 or 12% SDS-PAGE gels and blotted onto polyvinyl-idenedifluoride (PVDF) membranes. Membranes were treated with antibodies using standard Western blot techniques. The ECL Plus Detection Reagent (GE Healthcare, Chalfont St Giles. UK) was used for chemiluminescent detection, and membranes were analyzed using X-Omat LS films (Carestream, Rochester, NY) in a Curix 60 developer (Agfa, Mortsel, Belgium). Densitometry analysis was performed using the ImageLab Software (Biorad, Hercules, CA).
Antibodies used were: anti-LMP2 (Abcam, Cambridge, UK), anti-LMP7 (Abcam), anti-beta5 (Santa Cruz Biotechnology, Dallas, TX), anti-Tbp1 (Bethyl Laboratories, Montgomery, TX), anti-PSMD7 (Abcam), anti-PSMD11 (Novus Biologicals, Littleton, CO), anti-Alpha1–7 (Abcam), HRP conjugated anti-GAPDH (Cell Signaling, Cambridge, UK), and HRP conjugated anti-β-Actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies used were HRP conjugated goat anti-mouse IgG, and HRP conjugated goat anti-rabbit IgG (GE Healthcare).
Antibodies used were: anti-LMP2 (Abcam, Cambridge, UK), anti-LMP7 (Abcam), anti-beta5 (Santa Cruz Biotechnology, Dallas, TX), anti-Tbp1 (Bethyl Laboratories, Montgomery, TX), anti-PSMD7 (Abcam), anti-PSMD11 (Novus Biologicals, Littleton, CO), anti-Alpha1–7 (Abcam), HRP conjugated anti-GAPDH (Cell Signaling, Cambridge, UK), and HRP conjugated anti-β-Actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies used were HRP conjugated goat anti-mouse IgG, and HRP conjugated goat anti-rabbit IgG (GE Healthcare).
4
Profiling Acetylated Mitochondrial Proteins
5
Western Blot Analysis of Protein Samples
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Protein samples from both HEK293 cells and primary keratinocytes were lysed in RIPA buffer with protease/phosphatase inhibitor and denatured at 95 °C for 5 min or 50 °C for 10 min. Equal amounts of protein were loaded and electrophoresed on 8% SDS-polyacrylamide gels for 1.5 h at 120 V. The proteins were next transferred to a PVDF membrane in a cold room for 2 h at 120 V. After blocking for 1 h with BLOCK ACE (KAC, UKB80) at RT, the membrane was incubated with antibodies to detect target bands. The primary antibodies included mouse anti-FLAG (Sigma, F3165, 1:2000), rabbit anti-FLAG (Santa Cruz, sc-807, 1:1000), mouse anti-MYC (MBL, 1:1000), and HRP-conjugated anti-GAPDH (Cell Signaling, 3683, 1:1000). The secondary antibodies consisted of anti-mouse IgG (Cell Signaling, 7076, 1:10000) and anti-rabbit IgG (Cell Signaling, 7074, 1:10,000). All proteins were incubated with an ECL kit (Cytiva), and the blots were imaged using a LAS-3000 mini (Fujifilm). Quantification was conducted with ImageJ.
6
Protein Extraction and Western Blot Analysis
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Total protein extractions were performed as previously described [59 (link)] Nuclear/cytoplasmic protein fractionations were performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo, Waltham, MA, USA), according to manufacturer's instructions. Histone extraction [60 (link)] and immunopreciptiation [59 (link)] experiments were performed as previously described. Western blot analyses were performed according to standard protocols. Antibodies used were anti-ATM S1981p (Rockland, Philadelphia, PA, USA), anti-p53 DO-1, anti-p-p53 (Ser15), anti-ATM 2C1 (Santa Cruz), anti-RanBP9 (Abcam, Cambridge, MA, USA), anti-pChk2 (Thr68), anti-Chk2, anti-γH2AX, anti-H2A, anti-PARP, anti-GAPDH-HRP-conjugated, anti-β-actin (Cell Signaling Technology, Danvers, MA, USA) and anti-p84 (GeneTex, San Antonio, TX, USA). ImageQuant software (Biorad, Hercules, CA, USA) was used for the quantification of western blot data.
7
Antibody validation for SIRT1, PPARγ studies
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The antibodies used in this study are as follows: anti-SIRT1, cat. no. 07-131, Millipore; anti-PPARγ, cat. no. 81B8, Cell Signaling; anti-GAPDH-HRP conjugated, cat. no. GTX627408-01, GeneTex; Goat anti-Rabbit Alexa Fluor 594 conjugated, cat. no. R37117, Invitrogen; anti rabbit IgG-HRP conjugated, cat. no. 65-6120, Invitrogen. All purchased antibodies were validated for mammalian studies (as shown on the manufacturers’ websites). EX527 (E7034), FK866 (F8557), and NAD+ (N8535) were purchased from Sigma-Aldrich. β-NMN was purchased from Santa Cruz Biotechnology (sc-212376).
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