The total proteins were extracted, and the protein concentration was measured as described previously [8 (link)]. Total proteins (10 μg/lane) were separated by SDS-PAGE followed by transferring onto 0.22 μm PVDF membranes (Millipore, Bedford, MA, USA). Blot analysis was performed with an anti-IFN-γ antibody (Abcam, San Francisco, CA, USA, ab27919), an anti-TNF-β antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, SC-28345), an anti-IL-2 antibody (Abcam, ab193807), an anti-IL-4 antibody (Bio-Techne, MAB2469), an anti-IL-5 antibody (Santa Cruz Biotechnology, SC-8433), an anti-IL-6 antibody (Abcam, ab193853), and an anti-IL-10 antibody (Santa Cruz Biotechnology, SC-32815) at a dilution of 1:1000 to analyze expression of Th1 and Th2 cytokines respectively. Target proteins were detected by a Pro-light HRP chemiluminescence detection reagent (Tiangen Biotech, Beijing, China). An anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology, sc-20357, 1:1000) was used for normalization of sample loading. The intensity of blots were semi-quantified by Quantity One V452 (Bio-Rad Laboratories), and the values were calculated using GAPDH as an internal reference.
Pro light hrp chemiluminescence detection reagent
Manufactured by Tiangen Biotech
Sourced in China
The Pro-light HRP chemiluminescence detection reagent is a laboratory product designed for chemiluminescence detection. It is used to detect and quantify proteins that have been labeled with horseradish peroxidase (HRP) in Western blot or other immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one v452, by Bio-Rad (1 mentions)
Sc 20357, by Santa Cruz Biotechnology (1 mentions)
0.22 μm pvdf membrane, by Merck Group (1 mentions)
Ab203346, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Tiangen Biotech (1 mentions)
Bl001a, by Biosharp (1 mentions)
Bl003a, by Biosharp (1 mentions)
Sc 390328, by Abcam (1 mentions)
Sc 19641, by Santa Cruz Biotechnology (1 mentions)
Quantity one software v450, by Bio-Rad (1 mentions)
Bl504a, by Biosharp (1 mentions)
Gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Biosharp (1 mentions)
2 protocols using pro light hrp chemiluminescence detection reagent
1
Cytokine Expression Analysis by Western Blot
2
Western Blot Analysis of Metallothionein Proteins
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Total proteins from thymus, lymph node, spleen and liver samples were extracted with RIPA lysis buffer (Biosharp, BL504A) and then were quantified using a bicinchoninic acid protein assay kit (Tiangen Biotech). The proteins were separated using 12% sodium dodecyl sulphateÀpolyacrylamide gel electrophoresis, and then were transferred to 0Á22-lm polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% (w/v) fat-free milk. MT1, MT2 and CD4 were detected by a mouse anti-MT1 monoclonal antibody (Santa Cruz Biotechnology; sc-390328, 1:1000), a rabbit anti-MT2 polyclonal antibody (Abcam; ab203346, 1:1000; Cambridge, UK) and a mouse anti-CD4 monoclonal antibody (Santa Cruz Biotechnology; sc-19641, 1:1000), respectively. Secondary goat anti-mouse IgG-HRP (Biosharp, BL001A) and goat anti-rabbit IgG-HRP (Biosharp, BL003A) were diluted 1:10 000. Positive signals were detected by pro-light HRP chemiluminescence detection reagent (Tiangen Biotech). Sample loading was monitored with a GAPDH antibody (Santa Cruz Biotechnology; sc-20357), which was used at a dilution of 1:1000. Quantity One software (v450; Bio-Rad Laboratories, Hercules, CA) was used to quantify the blots.
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