Nuclear and cytoplasmic lysates containing equal amounts of protein (25 μg) were equally loaded on 12% SDS–polyacrylamide gel and transferred to PVDF membranes (Millipore) using a Mini-Protean 2 electrophoresis system (Bio-Rad Laboratories). After blocking with 5% skimmed milk in TBS plus 0.1% Tween-20, the membranes were incubated with NF-κB p65 rabbit polyclonal antibody (Proteintech) overnight at 4 °C followed by HRP-conjugated goat anti-rabbit IgG (Trans Gen Biotech). Detection was visualized using an ECL assay kit (Thermo Fisher Scientific, Inc.). Images were subsequently analyzed using Image J software to quantify the protein expression (National Institutes of Health, USA). All blots were repeated in triplicate.
Hrp conjugated goat anti rabbit igg
Manufactured by Transgene
Sourced in China
The HRP-conjugated goat anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. It is conjugated with horseradish peroxidase (HRP), which is an enzyme that can be used for colorimetric or chemiluminescent detection in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse igg, by Transgene (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Mini protean 2 electrophoresis system, by Bio-Rad (1 mentions)
Nf κb p65 rabbit polyclonal antibody, by Proteintech (1 mentions)
Ecl assay kit, by Thermo Fisher Scientific (1 mentions)
Ab126704, by Abcam (1 mentions)
Ab223868, by Abcam (1 mentions)
Ab155987, by Abcam (1 mentions)
Ab68477, by Abcam (1 mentions)
Easysee western blot kit, by Transgene (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Hrp conjugated 6 his tag antibody, by Abmart (1 mentions)
4 protocols using hrp conjugated goat anti rabbit igg
1
Western Blot Analysis of NF-κB p65
2
Western Blot Analysis of DNA Damage Markers
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Cells were directly lysed using 2 × protein lysing buffer (2% SDS, 5% β-mercaptoethanol, 0.5% sucrose and 0.2% bromophenol blue) at 4°C for 20 min, and then the lysates were heated in a metal bath at 98°C for 5 min. Cell lysates were separated on 4–12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked using 5% non-fat milk and immunoblotted using indicated antibodies. The protein signals were detected using a New-SUPER ECL Substrate Kit (Keygen Biotech, Nanjing, China). The primary antibodies used in this study targeted phospho-ATR (S428) (1:1000, abcam, ab178407), phospho-ATM (S1981) (1:1000, Cell Signaling Technology, #5883), phospho-p53 (S15) (1:1000, abcam, ab223868), phospho-histone H2AX (S139) (1:1000, Cell Signaling Technology, #9508), GCLM (1:1000, abcam, ab126704), HO-1 (1:1000, abcam, ab68477), phospho-p38 (T180/Y182) (1:500, Santa Cruz Biotechnology, sc-17852-R), phospho-Hsp27 (S82) (1:1000, abcam, ab155987), phospho-ATF2 (T71) (1:1000, abcam, ab32019) and Actin (1:3000, Bioss, #bs-0061R). The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10,000, TransGen Biotech, Beijing, China, #HS201-01) and HRP-conjugated goat anti-rabbit IgG (1:10,000, TransGen Biotech, Beijing, China, #HS101-01).
3
Analyzing MAPK and NF-κB Signaling in GM-DCs
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For analyzing the activation of MAPK and NF-κB signaling pathways, GM-DCs were treated with 102L.L and 103L.L for 30 min or 120 min and proteins were extracted by nucleoprotein and cytoplasmic protein extraction kit (Biorebo Biotech, China). 40 ng/mL LPS (Sigma-Aldrich, USA) was used as the positive control.
For detection of EGFP and OVA expression, the recombinant L.L with pNZ8149-penp-OVA (L.L-OVA) was cultured at 30°C to OD595nm of 0.2-0.6. Then 10 ng/ml nisin was added and cultured for 0 h, 9 h, 12 h and 24 h at 30°C. The recombinant L.L with pMG36e is auto-inducible. The recombinant L.L was harvested by centrifugation (12000 rpm, 10 min), and the proteins were obtained by liquid nitrogen grinding. The expression of related proteins was detected by Western blot, including EGFP (TransGen Biotech, China) and OVA (Elabscience, China). After incubation with HRP-conjugated goat anti-mouse IgG, or HRP-conjugated goat anti-rabbit IgG (TransGen Biotech, China), the target proteins were detected using EasySee Western blot kit (TransGen Biotech, China).
For detection of EGFP and OVA expression, the recombinant L.L with pNZ8149-penp-OVA (L.L-OVA) was cultured at 30°C to OD595nm of 0.2-0.6. Then 10 ng/ml nisin was added and cultured for 0 h, 9 h, 12 h and 24 h at 30°C. The recombinant L.L with pMG36e is auto-inducible. The recombinant L.L was harvested by centrifugation (12000 rpm, 10 min), and the proteins were obtained by liquid nitrogen grinding. The expression of related proteins was detected by Western blot, including EGFP (TransGen Biotech, China) and OVA (Elabscience, China). After incubation with HRP-conjugated goat anti-mouse IgG, or HRP-conjugated goat anti-rabbit IgG (TransGen Biotech, China), the target proteins were detected using EasySee Western blot kit (TransGen Biotech, China).
4
Detecting 6xHis-tagged Proteins and K. pneumoniae Glycans
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Western blotting was performed as described previously [40 (link)]. Horseradish peroxidase (HRP)-conjugated 6 × His tag antibody (Abmart Shanghai Co., Ltd., Shanghai, China) was used to detect proteins with a 6 × His tag. If there were more non-specific bands, the 6 × His antibody was incubated for 1 h with E. coli W3110 cell lysate prior to detection). The antibodies against K. pneumoniae serotype O1 and O2 were obtained by immunizing Japanese white rabbits with K. pneumoniae strain 355 and K. pneumoniae strain 041 whole bacteria respectively, and were used to detect the glycan fraction of glycoproteins. HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, Inc., Beijing, China) was used as the secondary antibody.
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