Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide electrophoresis gels and blotted on to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Non-specific binding was blocked with 5% skimmed milk in TBS. Blots were probed with either mouse horseradish peroxidase (HRP)-conjugated anti-β-actin monoclonal antibody (Kangchen Bio-tech, Shanghai, China), or rabbit anti-TBX5 polyclonal antibody (Abcam, Cambridge, UK) followed by HRP-conjugated goat anti-rabbit secondary antibody (Proteintech, Chicago, IL, USA). Proteins were detected using ECL reagents (Millipore).
Hrp conjugated goat anti rabbit secondary antibody
Manufactured by Proteintech
Sourced in United States
The HRP-conjugated goat anti-rabbit secondary antibody is a detection reagent designed to bind to and identify rabbit primary antibodies. It contains horseradish peroxidase (HRP) enzyme, which can be used for colorimetric or chemiluminescent detection in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
β actin, by Proteintech (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ecl reagent, by Merck Group (1 mentions)
Rabbit anti tbx5 polyclonal antibody, by Abcam (1 mentions)
Collagen 1 14695 1 ap, by Proteintech (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Rabbit polyclonal anti cith3, by Abcam (1 mentions)
Ripa buffer, by Sangon (1 mentions)
Nuclear and cytoplasmic extraction kit, by Boster Bio (1 mentions)
P p65 ser536, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
Phospho p65, by Cell Signaling Technology (1 mentions)
Myd88, by Boster Bio (1 mentions)
Nr4a1, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence detection kit, by Vazyme (1 mentions)
Gapdh, by Proteintech (1 mentions)
Bca protein assay kit, by Vazyme (1 mentions)
Penicillin streptomycin, by Procell (1 mentions)
12 protocols using hrp conjugated goat anti rabbit secondary antibody
1
Western Blot Analysis of TBX5
2
Immunohistochemical Analysis of Extracellular Matrix
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After deparaffinization and hydration, sections were blocked with 3% hydrogen peroxide, followed by treatments with 10% normal goat serum (Cat. No. 16210072; Gibco; Thermo Fisher Scientific), primary antibodies α-SMA (A2547, MilliporeSigma), Collagen I (14695–1-AP, Proteintech), Collagen II (15943–1-AP, Proteintech), and HRP-conjugated goat anti-rabbit secondary antibody (1:400; Cat. No. A32731; Invitrogen). After DAB treatment, microscopic images were taken.
3
Western Blot Analysis of Protein Expression
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Colon tissue was snap-frozen and homogenized in RIPA buffer (Sangon Biotech, Shanghai, China) containing protease inhibitors on ice. The homogenate was centrifuged at 12,000 rpm for 10 min at 4°C and the total protein was quantified using a BCA (Beyotime Biotechnology, Shanghai, China) assay. Equal amounts of proteins were mixed with loading buffer, loaded on gradient gels (12% SDS-PAGE), and then transferred to a PVDF membrane. The membrane was blocked with 5% milk in TBST and incubated with primary antibodies at 4°C overnight as listed: rabbit polyclonal anti-Cit-H3 (1:1000, ABCAm); rabbit polyclonal anti-GAPDH (1:2000, Servicebio); rabbit polyclonal anti-PAD4 (1:5000, Proteintech); rabbit polyclonal anti-MPO (1:3000, Proteintech); rabbit polyclonal anti-occludin (1:3000, Proteintech). After washing, the blot was finally incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5000, Proteintech) for 1h at room temperature. Bands were visualized using a chemiluminescent substrate (Sangon Biotech) and were quantified based on the endogenous GAPDH level using ImageJ software.
4
Analyzing NF-κB Pathway Activation in 293T Cells
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Six-well plates containing 293T cells were transfected with or without pEGFP-N1-NS1 (4 μg). After the cells were transfected with NS1 for 0, 1, 3, 6, 12, 24, 36, and 48 h, the cytoplasmic and nuclear protein were extracted using a nuclear and cytoplasmic extraction kit (Boster, China) according to the manufacturer's instructions [26 (link)]. The protein concentration was measured using BCA Protein Assay Kit (Boster), and the collected protein was stored at −80°C until use.
For the total protein treatment, the cells were collected at 0, 1, 3, 6, 12, 24, 36, or 48 h following plasmid transfection. The protein extracts were prepared from cells by suspension in lysis buffer containing protease inhibitor cocktail for 30 min on ice. Then 20 μg protein from each sample was used for western blot analysis as previously described [27 (link)]. The following rabbit polyclonal antibodies were used as primary antibody of the corresponding proteins: p65 with 1:1,000 dilution (Proteintech, China), phospho-p65 with 1:1000 dilution (p-p65 Ser536, Cell Signaling Technology, America), IκBα with 1:1,000 dilution (Proteintech), MyD88 with 1:100 dilution (Boster, China), TLR2 with 1:100 dilution (Boster),β-actin (1:1,000, Proteintech),and TBP (1:1,000, Proteintech). A HRP-conjugated goat anti-rabbit secondary antibody (1:5,000, Proteintech) were used in this study.
For the total protein treatment, the cells were collected at 0, 1, 3, 6, 12, 24, 36, or 48 h following plasmid transfection. The protein extracts were prepared from cells by suspension in lysis buffer containing protease inhibitor cocktail for 30 min on ice. Then 20 μg protein from each sample was used for western blot analysis as previously described [27 (link)]. The following rabbit polyclonal antibodies were used as primary antibody of the corresponding proteins: p65 with 1:1,000 dilution (Proteintech, China), phospho-p65 with 1:1000 dilution (p-p65 Ser536, Cell Signaling Technology, America), IκBα with 1:1,000 dilution (Proteintech), MyD88 with 1:100 dilution (Boster, China), TLR2 with 1:100 dilution (Boster),β-actin (1:1,000, Proteintech),and TBP (1:1,000, Proteintech). A HRP-conjugated goat anti-rabbit secondary antibody (1:5,000, Proteintech) were used in this study.
5
Quantitative Western Blot Analysis
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Total protein was extracted with PIPA buffer with protease inhibitors on ice for 20 min. After quantification of protein concentrations using Bicinchoninic Acid Assay, the proteins (20 μg) were separated to 10% SDS-PAGE followed by transferring to PVDF membranes (Millipore). Next, blocking with 5% non-fat milk for 1 h, the membranes were then cultured with primary antibodies overnight at 4°C, followed by incubating at room temperature for 1 h with a HRP-conjugated goat anti-rabbit secondary antibody (ProteinTech Group, dilution 1:2000, cat. no. SA00001-2). After washing with blocking solution, the membranes were determined by an enhanced chemiluminescence (ECL) system (Immun-Star™ HRP chemiluminescent detection kit, Bio-Rad Laboratories). β-Actin as an internal control was used in the present study. The semi-quantification of proteins was carried out using ImageJ software. The primary antibodies (dilution 1:1000, Cell Signaling Technology) include MDM2 (cat. no. 86934), Bcl-2 (cat. no. 3498), p53 (cat. no. 2527), Bax (cat. no. 14796), and β-actin (cat. no. 4970).
6
Western Blot Analysis of Chondrocyte Proteins
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Chondrocytes were lysed with RIPA buffer (pH 8.0) and extracted protein was quantified with BCA Protein Quantitation Kit. Thereafter, protein was isolated on SDS-PAGE gels as well as transferred onto PVDF membrane. The membrane was sealed lasting 1 h utilizing PBST plus 5% BSA and incubated by specific primary antibodies at 4°C overnight, containing NR4A1 (1:500; #25851-1-AP; Proteintech, Wuhan, China), NR4A2 (1:500; #66878-1-Ig; Proteintech), GAPDH (1:1000; #60004-1-Ig; Proteintech), MMP3 (1:500; #17873-1-AP; Proteintech), MMP13 (1:1000; #18165-1-AP; Proteintech) and Collagen II (1:800; #28459-1-AP; Proteintech). Afterwards, the membrane was incubated by HRP-conjugated goat anti-rabbit secondary antibody (1:2000; #SA00001-2; Proteintech) lasting 1 h. Protein bands were visualized utilizing ECL kits.
7
Western Blot Analysis of Oxidative Stress Markers
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Proteins from each group of cells were extracted using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime). The protein concentrations were determined using a BCA protein assay kit (Vazyme). Equal amounts (10 μg) of proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). Membranes were blocked with 5% skim milk buffer for 1 h at room temperature, and then incubated with primary antibodies against HO-1, Nrf2, or GAPDH (1:1000; Proteintech) overnight at 4 °C. The membranes were washed three times, and then incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:10000; Proteintech) for 1 h at room temperature. The protein bands were visualized with an enhanced chemiluminescence detection kit (Vazyme), and protein quantification was performed using ImageJ 1.8.0 software.
8
Dihydrexidine and Cisplatin Nephroprotective Effects
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Dihydrexidine hydrochloride and Cisplatin were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Minimum Essential Medium, penicillin-streptomycin, and fetal bovine serum (FBS) were purchased from Procell (Wuhan, China). Urea assay kit (BUN), creatinine (Cr) assay kit (sarcosine oxidase), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and reduced glutathione (GSH) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Kits and probes for MTT, Dihydroethidium (DHE), BCA, and H&E were purchased from Beyotime Biotech (Nantong, China). The following antibodies were used to detect the proteins of interest: Capsase 3 (Cat No. 66470-2-Ig, Proteintech, Rosemont, IL, USA), Capsase 9 (Cat No. 10380-1-AP, Proteintech), Bcl-2 (Cat No. 26593-1-AP, Proteintech), Bax (Cat No. 50599-2-Ig, Proteintech), GPX4 (67763-1-Ig, Proteintech), FSP1 (Cat No. 20886-1-AP, Proteintech), FTH1 (Cat No. 3998S, Cell Signaling), Phospho-YAP (Ser127) (Cat No. 13008S, Cell Signaling), YAP (Cat No. 4912S, Cell Signaling), Phospho-LATS1 (Cat No. 8654S, Cell Signaling), LATS1 (Cat No. 3477S, Cell Signaling), β-Actin (Cat No. 66009-1-Ig, Proteintech), and HRP-conjugated Goat Anti-Rabbit secondary antibody (Cat No. SA00001-2, Proteintech).
9
Immunohistochemical Staining of Tumor Sections
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Tumor cryosections or cells grown on chamber slides were fixed in 4% paraformaldehyde for 20 min at room temperature, and specimens were blocked with blocking serum at 37°C for 20 min. Then specimens were incubated with rabbit anti-human primary antibody (1:200, Proteintech) at 37°C for 2 h, and washed three times with PBS, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody (1:200, Proteintech) for 30 min at 37°C. Immunostaining was visualized using 3,3′-diaminobenzidine reagent followed by counterstaining with hematoxylin. Omitting primary antibody was considered as a negative control.
10
Pneumolysin Expression Regulation by Kaempferol
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The S. pneumoniae D39 cells cultured with kaempferol (0, 8, 16, 32 μg/mL) were collected and obtained an OD600 nm of 1.0. The samples were mixed with loading buffer and boiled at 100 °C for 10 min for immunoblotting analysis. Briefly, samples were separated by 12% SDS-PAGE and transferred onto the polyvinylidene fluoride (PVDF) membrane. After blocking, the membranes were incubated with anti-pneumolysin antibody (1:3000; Abcam) and HRP-conjugated goat anti-rabbit secondary antibodies (1:3000; Proteintech). Then, the membranes were visualized using an enhanced chemiluminescence substrate.
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