Total cell protein was extracted on ice using RIPA lysis buffer in the presence of freshly added protease and phosphatase inhibitors (Thermo), then quantified by the BCA method (Pierce). A total of 30 μg protein extract per lane was loaded onto a 14% SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Pierce). Nonspecific binding was blocked with 5% nonfat milk in PBS. The membrane was incubated with rat anti-IL-33 (R&D) or rabbit anti-phospho-STAT6 (Cell signaling) antibody overnight at 4°C. IRDye 800CW goat anti-rabbit IgG or goat anti-rat IgG (LI-COR) was used as secondary antibody, and rabbit anti-GAPDH antibody (Abcam) was used as an internal standard.
Rat anti il 33
Manufactured by R&D Systems
Rat anti-IL-33 is a monoclonal antibody that recognizes the interleukin-33 (IL-33) protein. IL-33 is a cytokine that plays a role in various inflammatory and immune processes. The antibody can be used for the detection and quantification of IL-33 in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Rabbit anti gapdh antibody, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Rabbit anti phospho stat6, by Cell Signaling Technology (1 mentions)
Complete mini, by Roche (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Rabbit anti α sma, by Abcam (1 mentions)
3 protocols using rat anti il 33
1
Western Blot for IL-33 and STAT6
2
Isolation and Western Blot of IL-33 in NOD Mice
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Isolated islets from female NOD mice of NOD Scid mice were digested in 100uL of digestion buffer (1X Cell Lysis Buffer (Cell Signaling Technology)) supplemented with protease inhibitors (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail, Roche) and 1mM PMSF (Cell Signaling Technology). 10 ug of the protein lysate were mixed with 4X Laemmli buffer (Bio-Rad) supplemented with 2-mercaptoethanol (Sigma-Aldrich) at a 3:1 (sample:buffer) ratio were denatured at 95°C for 5 minutes before loading onto a 15% SDS-PAGE gel, followed by a transfer onto a nitrocellulose membrane (Bio-Rad). Proteins were detected using Rat anti-IL-33 (R&D Systems) and Rabbit β-Actin (Cell Signaling Technology) antibodies followed by HRP-conjugated secondary antibodies (anti-Rabbit or anti-Rat antibodies (Cell Signaling Technology).
3
Liver Immunohistochemistry for IL-33 and α-SMA
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Immunohistochemistry was performed on formaldehyde-fixed, paraffin-embedded mouse livers. After hydration, liver sections were incubated with rat anti-IL-33 (R&D) or rabbit anti-α-SMA (Abcam) antibody for 1 hour at 37°C, and HRP or fluorescence conjugated secondary antibody (Abcam) was used to display the signals.
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