Horseradish peroxidase hrp conjugated secondary antibody

Pancreatic levels of Bcl-2, Bax and pancreatic duodenal homeobox-1 (PDX-1) protein were evaluated by western blot analysis. Total proteins were extracted with RIPA lysis buffer (Beyotime institute of Biotechnology, Haimen, China) supplemented with 1 mmol/L phenylmethanesulfonyl fluoride (PMSF, Sigma, MO, USA), followed by determination of protein concentrations by bicinchoninic acid (BCA) method (Beyotime institute of Biotechnology, Haimen, China). Then protein samples were subjected to SDS-polyacrylamide gel electrophoresis (Bio-Rad, CA, USA) and transferred to nitrocellulose membrane (Millipore, MA, USA). After blocking with 5 % skimmed milk in TBS containing 0.2 % Tween-20 (TBST), the membrane was incubated overnight at 4 °C with primary antibodies against Bcl-2, Bax, PDX-1 (Santa Cruz, CA, USA) and β-actin (Beyotime institute of Biotechnology, Haimen, China), followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibody (Beyotime institute of Biotechnology, Haimen, China) for 1 h at room temperature. Bound HRP was visualized with an enhanced chemiluminescence substrate (ECL, Beyotime institute of Biotechnology, Haimen, China) and protein bands were analyzed for integrated optical density (IOD) with Quantity One software (Bio-Rad, CA, USA). The levels of PDX-1, Bcl-2 and Bax were normalized to that of β-actin.

The tissue samples or cultured cells were lysed on ice for 30 min in RIPA buffer (Beyotime), supplemented with protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). After centrifugation (12,000g) at 4°C for 20 min, the supernatant was harvested and the protein concentration was determined by using BCA assay kit (Thermo Fisher Scientific). Protein extracts were separated by sodium dodecyl sulfate–polyacrylamide gel and electroblotted onto a nitrocellulose membrane. To block the non-specific binding sites, the membranes were incubated with 5% non-fat milk at room temperature for 60 min, and then probed with primary antibodies overnight at 4°C. After washing three times with Tris-buffered saline with 0.1% Tween-20 (TBST), the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime, Shanghai, China) at room temperature for 1 h. The specific proteins in the blots were visualized by using enhanced chemiluminescence (ECL, Millipore, Bredford, USA). The densities of the protein bands were quantified using Image J software (NIH, USA).

MKN-45 cells were incubated with the test compound at various concentrations for 24 h. After incubation, cells were harvested in PBS solution containing proteinase inhibitors and phosphatase inhibitors, and then sonicated. Whole cell lysates were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (Beyotime Biotechnology, Shanghai, China). The membrane was incubated with primary antibody followed by labelling with horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime Biotechnology, Shanghai, China). The blots were developed with enhanced chemiluminescence and visualised by an LAS4000 imager (Fuji Photo Film, Minato City, Japan).

The expression of in situ Hsp60 protein in pancreas was detected by immunohistochemistry, with the same protocol as the former [10 (link)]. Slides of the paraffin section of mouse pancreatic tissue dewaxed, hydrated, and heat-mediated antigen retrieval, and then blocked in 5% bovine serum albumin (BSA) (Sangon Biotech, Shanghai, China) for 1 h at room temperature. The slides were subsequently incubated with rabbit anti-Hsp60 antibody (1:600 diluted) (Enzo, Pennsylvania, USA) overnight at 4 °C, and horse radish peroxidase (HRP)-conjugated secondary antibody (Beyotime, Shanghai, China) for 2 h at room temperature. Finally, the slides were stained with diaminobenzidine (DAB) (Sangon Biotech, Shanghai, China) and hematoxylin.
Positive signals with brownish staining were observed under microscopy. Images were captured by Motic Med 6.0 (Motic Software Engineering Co. Ltd., Xiamen, China), and the optical density (OD) of the positive signals was measured semi-quantitatively by the software of Image J. Results were presented as a percentage of the NS1h group.

Cells of each group were collected and lysed by lysis buffer (Beyotime Biotechnology, China). The proteins were extracted and separated by SDS-PAGE, and were then transferred onto PVDF membranes. After blocking with 1% BSA for 1 h at room temperature (RT), the membranes were incubated with antibodies against GFP, Flag FAM289 (FAM92A1 Antibody), anti-Galectin-1, H3, ERK, NF-κB, DNMT1, DNMT3B, Galectin-1, SOX2, OCT4, CD133, C-Myc, GAPDH, anti-pERK1/2 and pNF-κB (The details of antibodies are provided in Additional file 6: Table S4) at 4 °C overnight. After washing 3 times with TBST, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000, Beyotime Biotechnology, China) at RT for 1 h. Then the membranes were washed 3 times with TBST and imaged with a gel imaging system (BIO-RAD, USA).