Astrocytes or brain tissues were digested in RIPA extraction buffer (Beyotime, China). Protein samples were separated by 8% SDS-PAGE and transferred onto PVDF (polyvinylidene difluoride) membranes (Millipore, United States) in tank transfer system (Bio-Rad, United States). Membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour, washed three times in TBST, and incubated overnight at 4°C with primary antibodies against C3 (1 : 1000 dilution, Abcam, USA) or β-actin (1 : 1000 dilution, Abcam, USA). After incubation with the HRP-conjugated goat anti-rabbit IgG secondary antibody (1 : 10000 dilution, Da-UN, China), immunoreactive bands were detected by enhanced chemiluminescence (Millipore, United States). The protein bands were quantitatively analysed using ImageJ software 1.52a.
Hrp conjugated goat anti rabbit igg secondary antibody
Manufactured by Merck Group
Sourced in China
The HRP-conjugated goat anti-rabbit IgG secondary antibody is a laboratory reagent used to detect the presence of rabbit primary antibodies in various immunoassays. It consists of a secondary antibody raised in goats that is conjugated to the enzyme horseradish peroxidase (HRP). This conjugate can be used to amplify and visualize the signal generated by the primary antibody-antigen interaction.
Automatically generated - may contain errors
Lab products found in correlation
Tank transfer system, by Bio-Rad (3 mentions)
Enhanced chemiluminescence, by Merck Group (3 mentions)
Ripa extraction buffer, by Beyotime (3 mentions)
β actin, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
Trans blot turbo blotting system, by Bio-Rad (1 mentions)
Ecl solution, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Rabbit anti inos, by Proteintech (1 mentions)
Rabbit anti cd206, by Proteintech (1 mentions)
Rabbit anti wnt5a, by Proteintech (1 mentions)
Rabbit anti gapdh, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Laminin b, by Abcam (1 mentions)
Anti gr, by Abcam (1 mentions)
Anti c jun, by Abcam (1 mentions)
Anti stat4, by Santa Cruz Biotechnology (1 mentions)
7 protocols using hrp conjugated goat anti rabbit igg secondary antibody
1
Western Blot Analysis of Astrocyte C3
2
Western Blot Analysis of 2A Cleavage
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To confirm the cleavage of 2A sites, we prepared two dishes of HEK293T cells on 100 mm culture dishes and infected one dish with 3 μl of HSV-hChR2-2A- eNpHR-2A-Venus. Then, 1 day after the infection, the cells were harvested and lysed in a 100-μl ice-cold lysis buffer (50 mM HEPES pH 8.0, 400 mM NaCl, 10% glycerol, 1% Triton X-100, 5 mM DTT) containing a protease inhibitor cocktail (11836153001, Roche). Total protein concentrations were measured by Bradford assay. Equal amounts of proteins (20 μg per lane) were separated on 7.5% sodium dodecyl sulfate-PAGE (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane using the Trans-Blot® Turbo™ Blotting System (Bio-Rad). After blocking with 5% non-fat dried milk (NFDM) in TNTX buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.2% Triton X-100) for 30 min at room temperature, the membrane was incubated with primary antibody against Venus (ab290, Abcam, 1:10,000) in 3% bovine serum albumin (BSA) overnight at 4°C. Then, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (12–348, Millipore, 1:2,000) for 1 h at room temperature. The blots were developed using ECL solution (RPN2232, GE Healthcare) and detected with ChemiDoc MP imaging system (Bio-Rad).
3
Western Blot Analysis of Neuroinflammatory Markers
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BV2 cells and mouse spinal cord L4‐5 segment tissues were digested in RIPA extraction buffer (Beyotime, China). Protein samples were separated by 10% SDS‐PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, United States) in tank transfer system (Bio‐Rad, United States). Membranes were blocked with 5% nonfat milk in buffer containing 0.1% Tween‐20 (TBST) for 1 h, washed three times in TBST, and incubated overnight at 4°C with primary antibodies including rabbit anti‐Wnt5a (1:1000 dilution; Proteintech; USA; 55,184‐1‐AP), rabbit anti‐CaMKII (1:1000 dilution; Proteintech; USA; 13,730‐1‐AP), rabbit anti‐NFAT (1:1000 dilution; Proteintech; US; 22,023‐1‐AP), rabbit anti‐GAPDH (1:10000 dilution; Proteintech; USA; 10,494‐1‐AP), rabbit anti‐iNOS (1:1000 dilution; Proteintech; US; 18,985‐1‐AP), and rabbit anti‐CD206(1:1000 dilution; Proteintech; US; 18,704‐1‐AP). After incubation with the HRP‐conjugated goat anti‐rabbit IgG secondary antibody (1:10000 dilution, Da‐UN, China), immunoreactive bands were detected by enhanced chemiluminescence (Millipore, United States). The protein bands were quantitatively analyzed using ImageJ software 1.8.0 (National Institutes of Health, Bethesda, MA, USA).
4
Wnt5a-ROR2-JNK Signaling in Spinal Cord
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Mouse spinal cord L4-5 segment tissues were digested in RIPA extraction buffer (Beyotime, China). Protein samples were separated by 10% SDS-PAGE and transferred onto PVDF (polyvinylidene difluoride) membranes (Millipore, United States) in tank transfer system (Bio-Rad, United States). Membranes were blocked with 5% non-fat milk in buffer containing 0.1% Tween-20 (TBST) for 1 h, washed three times in TBST, and incubated overnight at 4℃ with primary antibodies including rabbit anti-Wnt5a (1:1000 dilution; Proteintech; USA; 55184-1-AP), mouse ROR2 Monoclonal antibody(1:1000 dilution; Proteintech; USA; 67906-1-Ig), mouse JNK Monoclonal antibody (1:1000 dilution; Proteintech; USA; 66210-1-Ig), rabbit Phospho-JNK Recombinant antibody (1:1000 dilution; Proteintech; USA; 80024-1-RR), rabbit anti-iNOS (1:1000 dilution; Proteintech; US; 18985-1-AP), rabbit anti-CD206(1:1000 dilution; Proteintech; US; 18704-1-AP), mouse Beta Actin Monoclonal antibody (1:10000 dilution; Proteintech; USA; 66009-1-Ig). After incubation with the HRP-conjugated goat anti-rabbit IgG secondary antibody (1:10,000 dilution, Da-UN, China), immunoreactive bands were detected by enhanced chemiluminescence (Millipore, United States). The protein bands were quantitatively analysed using ImageJ software 1.8.0 (National Institutes of Health, Bethesda, MA, USA).
5
Western Blot Analysis of NK92 Cells
6
Kidney Protein Expression Analysis
7
Protein Expression Analysis by Western Blot
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Total protein was extracted with RIPA lysis buffer, and its concentration was measured using a BCA Protein Assay Kit (KGP902; KeyGen Biotech, Nanjing, China). Proteins were separated by 10% SDS-PAGE electrophoresis and transferred to a nitrocellulose filter membrane. The membranes were incubated with blocking buffer consisting of 5% nonfat dry milk dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBST), for 60 min at room temperature. After washing the membranes with TBST, they were incubated at 4°C overnight with the following primary antibodies: anti-MMP-2, anti-MMP-9, anti-E-cad, anti-VEGF, anti-Vimentin, anti-N-cad, or anti-GAPDH polyclonal antibodies (diluted 1 : 2000 in blocking solution; Sigma-Aldrich). An enhanced chemiluminescence reagent (KeyGen, Nanjing, China) was added after incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (1 : 5000 in blocking solution; Sigma-Aldrich) for 1 h at room temperature. A Bio-Rad gel imaging system was used for protein band visualization, GAPDH was used as an internal reference, and the relative protein expression level was calculated by the ratio of the gray value of the target protein to that of GAPDH.
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