Horseradish peroxidase hrp conjugated secondary antibody

Western blotting was performed according to a standard protocol. Total cell or tissue lysates were extracted using 2% SDS lysis buffer (Applygen, Beijing), and 30 μg of total proteins were separated on 12% (v/v) SDS-PAGE gels. After electrophoresis, the proteins were transferred onto nitrocellulose membranes (Millipore, Ireland) and the membrane was incubated with the anti-CXCL5 antibody (1:500, ab9802, Abcam) and anti-GAPDH antibody (1:1000, H-12; Santa Cruz Biotechnology) at 4 ℃ overnight. After washing three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000, Zsbio, Beijing) for 1 h at room temperature. Finally, the membranes were visualized using an ECL Kit (Applygen, Beijing).

Treated tissues and cells were lysed by RIPA Lysis Buffer (Beyotime, Shanghai, China), and the protein concentration was measured by BCA protein assay kit (Beyotime, Shanghai, China). The immunoblotting was performed as previously described [47 (link)]. Briefly, the protein was separated in 12% SDS denatured polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% skim milk in Tris-buffered saline, pH 7.4, containing 0.1% Tween 20, at room temperature for 1 h, and were incubated with appropriate antibodies at 4°C overnight respectively. Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000, ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. Finally, the protein of interest was visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).

Western blot was performed using the documented standard methods as we have done before [9 ]. Retinas (four eyes from four mice from each group) were dissected, immersed and homogenized in 0.1% triton X-100 extraction buffer (Solarbio, Beijing, China) containing phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT), then the lysates were centrifuged to remove insoluble material, after which total retinal protein was extracted. Protein concentration was measured by a protein assay (Bradford Protein Assay; Bio-Rad, Hercules, CA, USA), and adjusted to allow equal total protein loading on the gels. Proteins were transfer to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) using standard electroblotting procedures. Then membranes were blocked with 5% skim milk and then incubated with cyclin D1(1:500 in 5% BSA) (AC853, Beyotime Biotechnology, Jiangsu, China), or β-actin (1:5000 in 5% BSA) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibodies at 4 °C overnight. Then membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (ZSGB-BIO, Beijing, China) at 37 °C for 1 h, and visualized by the ChemiDoc™ MP System (Bio-Rad, Hercules, CA, USA). Protein bands were quantified using Image-Pro plus 6.0 analysis software.

Western blotting was carried out as described previously.^{45 (link)} Total protein was extracted from the cultured cells using RIPA lysis buffer (Beyotime, Nanjing, China), which was supplemented with a protease inhibitor cocktail according to the manufacturer’s instructions. The protein mixtures were centrifuged to remove cellular debris. The extracted proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Amersham, Little Chalfont, UK). The PVDF membranes were blocked for 2 h at room temperature using 5% non-fat milk (BD Biosciences, San Jose, CA). The membranes were incubated at 4 °C overnight with the following primary antibodies: anti-OCN (1:1 000; Boster Biological Technology, Wuhan, China), anti-BSP (1:1 000; Boster Biological Technology), anti-ALP (1:1000; Proteintech Group, Wuhan, China), anti-RUNX2 (1:1 000; Proteintech Group), and anti-GAPDH (1:1 000; Boster Biological Technology). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (ZSGB-Bio) for 1 h at room temperature. The immunoreactive bands were visualized using an ECL chemiluminescence reagent (Solarbio). The relative intensities were analyzed and compared to their respective controls using ImageJ software (NIH, USA).

Cells were lysed in RIPA buffer (50 mmol/L Tris [pH 8], 250 mmol/L NaCl, 0.05% sodium dodecyl sulphate, 0.5% deoxycholate, 1% NP‐40). Proteins were separated on a 10% Bis‐Tris protein gel (1.0 mm) and transferred to nitrocellulose membrane (Bio‐Rad). Antibodies used were p‐p38 MAPK (4631; CST), p38 MAPK (8690; CST), p‐JNK (4668; CST), JNK (9252; CST), p‐Erk1/2 (4370; CST), Erk1/2 (4695; CST) and α‐tubulin (41517; SAB). Horseradish peroxidase (HRP)‐conjugated secondary antibodies were purchased from ZSGB‐BIO. The membranes were detected with a chemiluminescent reagent kit. α‐Tubulin served as an internal control. For image analysis, the films were scanned with a GS‐700 imaging densitometer (Bio‐Rad). Relative protein expression levels were measured using ImageJ software (National Institute of Health).