For Western blot, 2 × 105 cells were seeded in six-well tissue culture plates, exposed to the indicated treatment, then were scraped and lysed with RIPA lysis buffer (Beyotime Technology, Shanghai, China) or the nuclear and cytoplasmic protein extraction kit (P0027, Beyotime Technology, Shanghai, China) containing protease and phosphatase inhibitors (Pierce Protease and Phosphatase Inhibitor Mini Tables). After running in the 5–15% SDS-PAGE gels and transferred onto NC membrane (Life Technologies), membranes were blocked for 60 min at room temperature with 5% nonfat milk in TBS with 0.1% Tween 20 and incubated overnight at 4°C with the primary antibodies. The primary antibodies incubated with horseradish peroxidase-conjugated secondary antibodies (ABclonal Technology, Wuhan, China) for 1 h at room temperature and immunoreactive bands were detected using an ECL kit. The density of the immunoreactive bands was analyzed using ImageJ software.
Horseradish peroxidase conjugated secondary antibody
Manufactured by ABclonal
Sourced in China
Horseradish peroxidase-conjugated secondary antibodies are a type of laboratory reagent used in various immunoassay techniques. They consist of a secondary antibody that is conjugated with the enzyme horseradish peroxidase. This enzyme can be utilized to catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and quantification of target analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ripa lysis buffer, by Keygen Biotech (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Pgc 1α, by Proteintech (2 mentions)
Nf κb, by Proteintech (2 mentions)
Ampkα, by Proteintech (2 mentions)
P nf κb, by ABclonal (2 mentions)
Gapdh, by ABclonal (2 mentions)
Nc membrane, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Quickblocktm western buffer, by Beyotime (1 mentions)
Beyoecl plus, by Beyotime (1 mentions)
Calnexin, by ABclonal (1 mentions)
Bradford assay, by Thermo Fisher Scientific (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
18 protocols using horseradish peroxidase conjugated secondary antibody
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Extracellular Vesicle Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein concentration was measured by a BCA Protein Assay Kit (Beyotime; Shanghai, China; Cat. No. P0010S). Total proteins were dissolved using 6–12% SDS-PAGE and transferred to the polyvinylidene difluoride membranes (Millipore, Massachusetts, USA). The membranes were then blocked with QuickBlockTM western buffer (Beyotime; Cat. No. P0231) for 20 min at room temperature and incubated with primary antibody including CD63 (Abcam), CD9 (Diagbio; Hangzhou, China; Cat. No. db919), ALIX (Diagbio; Hangzhou, China; Cat. No. db3856), HSP70 (Diagbio; Hangzhou, China; Cat. No. db2396), Calnexin (Abclonal, Wuhan, China; Cat. No. A0803), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Diagbio; Hangzhou, China; Cat. No. db106), and GAS1 (absin, Shanghai, China; Cat. No. abs141177) at 4 °C overnight. Following, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Abclonal, Wuhan, China; 1:3000) at room temperature for 2 h. The blots were detected using BeyoECL Plus (Beyotime; Shanghai, China; Cat. No. P0018S).
3
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The collected cell or tissue samples were lysed for protein extraction, and the protein concentrations were measured using the Bradford assay (Thermo, Hercules, CA, USA). Protein samples were subjected to standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. After blocking in 5% non-fat milk for 1 h, the membranes were incubated with primary antibody against Bcl-2, Bax, Beclin1, p62, FNDC5 (Cell Signaling Technology, Boston, USA), and glyceraldehyde-phosphate dehydrogenase (GAPDH) (Santa Cruz, CA, USA) at 4 °C for 12 h. After washing with buffer, the membranes were incubated with the corresponding horseradish peroxidase‐conjugated secondary antibodies (Abclonal, Wuhan, China) for 1 h. Protein bands were visualized using the BioRad imaging system (Bio‐Rad, CA, USA).
4
Western Blot Analysis of Necroptosis Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cold radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology) was used to lyse the H9c2 cells according to standard protocols and the Pierce BCA Protein Assay kit (Beyotime Institute of Biotechnology) was used to measure protein concentration. A variety of SDS-PAGE (8–12%) was used to separate 30 mg protein per well. Polyvinylidene difluoride membranes were used to transfer the protein from the gels. Next the membranes were blocked using 5% skimmed milk at room temperature for 2 h and then the membranes were incubated with the following primary antibodies (all 1:1,000) overnight at 4°C: Receptor-interacting protein kinase 3 (RIP3; cat. no. A5431), mixed-lineage kinase domain-like protein (MLKL; cat. no. A17312) both from ABclonal, phosphate-NF-κB p65 (cat. no. sc-136548), NF-κB p65 (cat. no. sc-8008) both from Santa Cruz Biotechnology, Inc., TLR4 (cat. no. A5258; ABclonal) and β-actin (cat. no. AC026) both from ABclonal. The membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (ABclonal) for 2 h and the positive signals were detected at room temperature. Enhanced chemiluminescence (ECL) western blotting substrate (Thermo Fisher Scientific, Inc.) was used to visualize the protein bands, and ImageJ 1.47 software (National Institutes of Health) was used to semi-quantify the intensity.
5
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared in RIPA lysis buffer with the addition of a mammalian protease inhibitor cocktail (Sigma) and phosphatase inhibitor. Protein concentration was measured by BCA protein assay kit. Equal amounts of cell lysates were electrophoresed on SDS-polyacrylamide gels and transferred by electroblotting onto a polyvinylidene fluoride (PVDF) membrane. The following primary antibodies were used: mouse SHP1 (1:1000; abs158291, ABsin), and rabbit PKM2 (1:1000; 4053, CST Technology), p-PKM2 (1:1000; 3827, CST Technology), LDHB (1:1000; A5131, ABclonal), LDHA (1:1000; A1146, ABclonal). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000; ABclonal Technology). After one-hour incubation, membranes were washed three times with TBST, and then visualized by immunoblotting.
6
Analyzing ADAM23 Protein Expression in Psoriatic Skin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Psoriasis skin of mice lesions were harvested and lysed on ice with RIPA buffer. Protein samples were analyzed using 10% or 12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% non-fat milk for 1 h at room temperature (25 °C), washed three times in TBST, and co-incubated with the primary antibody overnight at 4 °C. The membranes were washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000; ABclonal, Wuhan, China) for 1 h at room temperature. The bands were detected using a western ECL blotting substrate according to the manufacturer’s protocol. The following primary antibodies were used: anti-ADAM23 polyclonal antibody (diluted 1:1000; Bioss) and anti-GAPDH antibody (1:1000; AC001; ABclonal). Densitometric analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The Western blot data from mice are expressed as means ± the standard deviation. Comparisons were performed using an unpaired t-test. Differences were considered statistically significant at two-tailed p-values < 0.05. GraphPad Prism V8.4.3 software (Prism 8 for macOS; San Diego, CA, USA) was used for the statistical analyses and to generate graphs.
7
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or tissues were lysed in a RIPA buffer (Solarbio, Beijing, China) supplemented with PMSF. After homogenizing for 5 min, the lysates were centrifuged at 13,000× g for 30 min. Protein concentration was measured using BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins in the lysates were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1 h and incubated with primary antibodies including those against PCSK6 (1:4000, Abcam, Cambridge, UK), P16 (1:1000, Abcam, Cambridge, UK), P21 (1:1000, Abcam, Cambridge, UK), DDIT3 (1:1000, CST, Danvers, MA, USA), and GAPDH (1:10,000, Proteintech, Wuhan, Hubei, China) at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies were used for visualization (ABclonal, Wuhan, Hubei, China). The protein bands were detected by SuperSignal ECL detection kit (Beyotime, Shanghai, China). Quantification of band intensities, normalized to GAPDH, was carried out using ImageJ (NIH, Bethesda, MD, USA).
8
Testicular Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins of the mouse testicular tissues were extracted using a universal protein extraction lysis buffer (Bioteke, Winooski, USA) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). The denatured proteins were separated on 10% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, USA). Then, the membrane was blocked for 0.5 h at room temperature and incubated overnight at 4°C with the following primary antibodies: anti-Cep112 (1:1000; ThermoFisher), anti-Na/K-ATPase (1:500; Abclonal), anti-Spata6 (1:1000; ThermoFisher), anti-Wdr60 (1:500; ThermoFisher), anti-Akap3 (1:1000; Abcam), and anti-Tubulin (1:500; Abclonal) antibodies. After being washed with 0.3% TBST, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibodies (1:5000; Abclonal) for 1 h at room temperature. Then the blots were visualized with the gel imaging system (Beijing Liuyi Biotechnology, Beijing, China).
9
Antibody Procurement for Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit monoclonal antibodies against ITGB3 (#4702) was purchased from Cell Signaling Technology. Mouse monoclonal antibodies against β-actin (#AC010), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#AC002) and horseradish peroxidase-conjugated secondary antibodies were purchased from ABclonal Technology, China.
10
Chondrocyte Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chondrocytes were lysed with RIPA lysis buffer (KeyGEN) supplemented with protease inhibitors. Total protein was collected and denatured. After a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were transferred to a polyvinylidene di uoride membrane (Millipore). Blots were further probed with primary antibodies (listed in Supplementary Table 3) and horseradish peroxidaseconjugated secondary antibodies (ABclonal). After washing, the membrane was visualized using an enhanced chemiluminescence kit (Ncm Biotech).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!