Proteins were extracted using RIPA total protein lysate (cat. no. P0013C, Beyotime) according to the manufacturer's instructions. Proteins from each sample were separated by electrophoresis on 10% SDS-PAGE gels and transferred onto PVDF membranes (cat. no. FFP39, Beyotime). After incubation with the blocking solution (cat. no. P0252, Beyotime) for 1 h at room temperature, the PVDF membranes were separately incubated with primary antibodies (1:1000; Abcam, Cambridge, UK) at 4 °C overnight. After the blots were washed, they were incubated with HRP-conjugated goat anti-rabbit IgG H&L secondary antibody (1:2000; cat.no.ab6721, Abcam) for 1.5 h and detected using ECL western reagent (cat. no. P0018S, Beyotime) on a chemiluminescent imaging system (ChemiDoc MP, Bio-Rad, CA, USA).
Hrp conjugated goat anti rabbit igg h l secondary antibody
Manufactured by Abcam
Sourced in China, United States
HRP-conjugated goat anti-rabbit IgG H&L secondary antibody is a laboratory reagent used to detect and quantify rabbit primary antibodies in various immunoassays. It consists of goat-derived antibodies specific to the heavy and light chains of rabbit immunoglobulin G (IgG), conjugated to the enzyme horseradish peroxidase (HRP).
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp, by Bio-Rad (1 mentions)
Gtx55114, by GeneTex (1 mentions)
Ecl reagent, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Bca protein quantification kit, by Yeasen (1 mentions)
Ab32351, by Abcam (1 mentions)
Ab2324, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab109520, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Imagej software, by Bio-Rad (1 mentions)
Goat serum, by ZSGB-BIO (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Cell lysis buffer for western and ip, by Beyotime (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
5 protocols using hrp conjugated goat anti rabbit igg h l secondary antibody
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of IL1B and NF-kB
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The total protein in the cells was extracted using RIPA lysis buffer and quantified using the BCA Protein Quantification Kit (Yeasen Biotechnology Co., Ltd., Shanghai, China). Medium amounts of proteins were prepared, separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane. The membranes were blocked in 5% bovine serum albumin solution at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies specific for IL1B (1:1000, NBP1-42767, Novus Biological Inc., Littleton, CO, USA), phospho–NF–κB p65 (Ser536) (1:2000, GTX55114, GeneTex), NF-κB p65 (1:2000, #8242, Cell Signaling Technologies, Beverly, MA, USA), and GAPDH (1:1000, #2118, Cell Signaling Technologies). The membranes were then incubated with HRP-conjugated goat anti-rabbit IgG H&L secondary antibody (1:5000, ab6721, Abcam) for 1 h at room temperature and developed using an ECL reagent (Abcam). ImageJ software was used to quantify protein band densities. The densities of individual protein bands were normalized to those of the corresponding GAPDH band.
3
Western Blot Analysis of Apoptosis Markers
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GC cells were lyzed with the Radioimmunoprecipitation Assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor (Roche, USA). The protein concentration was determined using the bicinchoninic acid (BCA) Protein Assay Kit (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA, USA). A total of 20 μg protein was loaded, separated by 15% SDS-PAGE, and transferred onto the polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk at room temperature (RT) for 1 h, the membranes were probed with primary antibody against DEK (1:1000 dilution; ab221545; Abcam, Shanghai, China), cleaved caspase-3 (1:1000 dilution; ab32351; Abcam, Shanghai, China), cleaved caspase-9 (1:1000 dilution; ab2324; Abcam, Shanghai, China), p21 (1:2000 dilution; ab109520; Abcam, Shanghai, China) or GAPDH (1:2000 dilution; ab181602; Abcam, Shanghai, China) overnight at 4ºC. After washing twice with PBST, the membranes were incubated with HRP-conjugated Goat anti-Rabbit IgG H&L secondary antibody (1:3000 dilution; ab205718, Abcam, Shanghai, China) for 1 h at RT. The signals were developed using the Enhanced Chemiluminescence kit (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and analyzed with the Image J software (Version 5.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
4
Immunohistochemical Analysis of WDR45B in HCC
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The HCC tissue microarray was made by formalin-fixed paraffin-embedded (FFPE) specimens originating from 56 patients with primary HCC date from January 2021 to December 2021. The FFPE specimens of 3 patients with hepatic contusion were used as normal control. All the paraffin-embedded specimens were provided by the Pathology Department of Zhongnan Hospital of Wuhan University. The paraffin sections were dried at 60 °C for 1 h and dewaxed in xylene and ethanol. Citrate buffer was used to repair antigen, and then the sections were blocked by 3% hydrogen peroxide for 10 min and 5% goat serum (ZSGB-Bio, Beijing, China) for 1 h. Afterward, sections were incubated at 4 °C overnight with the WDR45B primary antibody (1:200, Signalway Antibody). On the following day, the sections were incubated with HRP-conjugated Goat Anti-Rabbit IgG H&L secondary antibody (abcam, Cambridge, MA, USA), and the DAB (Dako Diagnostics AG, Baar, Switzerland) was stained in appropriate time for microscopic examination. Washing with PBS three times was included between all processes. For imaging, the sections were scanned by an Olympus BX51 microscope equipped with a DP74 digital camera. Both the staining intensity and degree were assessed in semi-quantitative analysis by ImagePro Plus 6.0.
5
Western Blot Analysis of YAP1 in Kidney Cancer Cells
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Total protein was extracted from Caki-1 and A498 cells using a Cell lysis buffer for Western and IP (Beyotime Institute of Biotechnology) following the manufacturer's protocol. The protein samples were quantified using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). A total of 30 µg protein/sample were separated by 10% SDS-PAGE, and subsequently transferred onto a nitrocellulose membrane (MilliporeSigma). The membrane was blocked with 5% skimmed milk in TBS-Tween-20 (0.1%) buffer for 1 h at room temperature. Subsequently, the membrane was incubated with primary antibodies against YAP1 (1:600; cat. no. ab52771) and GAPDH (1:4,000; cat. no. ab8245) (both from Abcam) overnight at 4˚C. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG H&L secondary antibody (1:20,000; cat. no. ab6721; Abcam) for 1 h at room temperature. Finally, the protein bands were visualized with an ECL kit (GE Healthcare) and analyzed using ImageJ v1.8.0 software (National Institutes of Health).
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