Cultured IgM+ B cells were collected and lysed with RIPA lysis buffer (Beyotime Biotechnology). Total cell lysates were mixed with loading buffer, boiled for 10 min, and then subjected to SDS-PAGE electrophoresis. Proteins were transferred onto PVDF membranes (GE Healthcare). After blocking with 5% skim milk, PVDF membranes were incubated with different primary antibodies overnight at 4°C, followed by incubation with peroxidase-conjugated goat anti-Rabbit IgG at room temperature for 1 h. Bands were visualized using chemiluminescence detection reagents (Advansta). All antibodies used in this study were purchased from Cell Signaling Technology, except for the p-STAT3(S727) monoclonal antibody, which was from Abcam.
Chemiluminescence detection reagents
Manufactured by Advansta
Sourced in United States
Chemiluminescence detection reagents are a set of chemical compounds that emit light upon a specific chemical reaction. These reagents are used in various laboratory techniques, such as Western blotting and ELISA, to detect and quantify target proteins or analytes. The core function of these reagents is to generate a luminescent signal that can be measured and analyzed to determine the presence and abundance of the target molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Anti rab igg secondary rabbit antibody, by OriGene (2 mentions)
Goat anti rat igg, by OriGene (2 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Occludin, by Proteintech (1 mentions)
Ve cadherin, by Cell Signaling Technology (1 mentions)
Gapdh, by GeneTex (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti hdac1 antibody, by Cell Signaling Technology (1 mentions)
5 protocols using chemiluminescence detection reagents
1
Immunoblotting of IgM+ B Cells
2
Western Blot Analysis of GP2 Protein
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Total proteins were prepared from tissue samples of the colon using RIPA lysis buffer (Beyotime, Shanghai, China) with protease inhibitor. A total of 20 µg of protein was loaded in each lane for SDS-PAGE, transferred, and immunoblotted overnight at 4 °C with a primary antibody against GP2 (1:1000, MBL, Nagoya, Japan) and GAPDH (1:5000, Proteintech Group, Rosemont, IL, USA), and Goat anti-Rat IgG (OriGene Technologies, Rockville, MD, USA). Next, the membrane was incubated with a 1:5000 dilution of anti-Rab IgG secondary rabbit antibody (OriGene Technologies, Beijing, China). Finally, the immunoblots were developed with chemiluminescence detection reagents (Advansta, San Jose, CA, USA) [20 (link),21 (link)].
3
Western Blot Analysis of Cell Junctions
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First, protein samples from cells were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer. The protein samples were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electroblotting onto polyvinylidene fluoride (PVDF) membranes. The PVDF membrane was connected to the positive electrode on one side (red) and the gel to the negative electrode on the other side (black). The membranes were then probed with monoclonal antibodies against ZO-1 (Proteintech group), polyclonal antibodies against VE-cadherin (Cell Signaling Technology), occludin (Proteintech), and GAPDH (GeneTex). Finally, the protein bands were visualized using chemiluminescence detection reagents (Advansta), and densitometric analysis was conducted using imaging processing software (Image J 1.8.0_172).
4
GP2 Protein Expression in Colon Tissue
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Total proteins were prepared from tissue samples of the colon using RIPA lysis buffer (Beyotime, China) with protease inhibitor. A total of 20 µg of protein were loaded in each well for SDS-PAGE, transferred, and immunoblotted overnight at 4°C with the primary antibody against GP2 (1:1000, MBL D278-3, Japan) and GAPDH (1:5000, Proteintech Group, U.S.). Goat anti-Rat IgG (OriGene Technologies, U.S.) and anti-Rab IgG secondary rabbit antibody (OriGene Technologies, U.S.) were diluted at 1:5000 for incubation with the membrane. Finally, the immunoblots were developed with chemiluminescence detection reagents (Advansta, U.S.) [20, 21] .
5
SDS-PAGE Western Blot Analysis
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Proteins were separated with 8.5% SDS-PAGE and transferred to polyvinylidene difluoride membrane. After blocking with 5% nonfat dry milk containing TBS-0.1% Tween20 (TBST) buffer, membrane was probed with anti-p65 antibody (1:5,000, C-20X; Santa Cruz, Santa Cruz, Calif), anti-CCRP antibody (1:5,000, generated as previously described, 7), anti-HSP90 antibody (1:1,000, 610419; BD Transduction Laboratories, San Jose, Calif), anti-HDAC1 antibody (1:1,000, #2062; Cell Signaling Technology, Beverly, Mass), or anti-βACTIN antibody (1:1,000, C4; Santa Cruz) in the 5% milk or 5% BSA containing TBST buffer for overnight at 4°C and then with proper secondary HRP-conjugated antibody (1:5,000, sc-2314 and sc-2004; Santa Cruz) for 1 h at room temperature. Protein bands on membrane were visualized using chemiluminescence detection reagents with various sensitivity to obtain desired band intensity (Advansta, Menlo Park, Calif).
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