Cell lysates and culture supernatants were incubated with cell lysis buffer (20 mM Tris HCl pH 7.4, 200 mM NaCl, 1% NP-40) and denatured in laemmli buffer. Subsequently the protein samples were boiled at 95°C for 10 min and separated by SDS-PAGE. Separated proteins were transferred to PVDF membranes. Blocking, incubation with antibody and washing of the membrane were done in PBS supplemented with 0.05% Tween-20 (v/v) and 3% (w/v) non-fat dry milk. Immunoblots were incubated overnight with primary antibodies against caspase-1 (AG-20B-0042-C100, Adipogen), Nlrp3 (AG-20B-0014-C100, Adipogen), ASC (AG-25B-0006, Adipogen), IL-1β (GTX74034, Genetex), IL-18 (5180R-100, Biovision), IkBα (9242S, Cell Signaling), Phospho-IkBα (2859S, Ser32) (Cell Signaling), β-actin (NB600-501H, Novus Biologicals) and A20 (sc-166692, Santa Cruz Biotechnology). Horseradish peroxidase-conjugated goat anti-mouse (115-035-146, Jackson Immunoresearch Laboratories) or anti-rabbit secondary antibody (111-035-144, Jackson Immunoresearch Laboratories) was used to detect proteins by enhanced chemiluminescence (Thermo Scientific).
Sc 166692
Manufactured by Santa Cruz Biotechnology
Sc-166692 is a laboratory equipment designed for scientific research applications. It is intended to be used as a tool for conducting various experiments and analyses within a controlled laboratory environment. The core function of this product is to provide a reliable and precise means of handling and processing samples or substances during the course of scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Ag 20b 0014 c100, by Adipogen (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Gtx74034, by GeneTex (1 mentions)
Ag 25b 0006, by Adipogen (1 mentions)
Ag 20b 0042 c100, by Adipogen (1 mentions)
Sc 22165, by Santa Cruz Biotechnology (1 mentions)
β actin hrp, by Santa Cruz Biotechnology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
2 protocols using sc 166692
1
Western Blot Analysis of Inflammasome Proteins
2
Western Blot Analysis of A20 and Caspase-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equally seeded cell cultures were lysed in lysis buffer (20 mM Tris HCl (pH 7.4), 200 mM NaCl, 1% Nonidet P-40) and were denatured in 4x Laemmli buffer. Proteins were separated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane followed by incubation overnight at 4°C with primary antibody against A20 (1:1000, SC- 166692, Santa Cruz Biotechnology), caspase-1 p20 (1:1000, SC-22165, Santa Cruz) and HRP-conjugated secondary goat anti-rabbit (1:2500, DAKO) in Tris-buffered Saline supplemented with 0.05% Tween-20 and 5% nonfat dry milk. Detection was performed with chemiluminescence (Western Lightning Plus-ECL, PerkinElmer) using an Amersham Imager 600 (GE Healthcare). After imaging, blots were incubated with directly labeled primary antibody β-actin-HRP (1:10000, Santa Cruz) for 15 minutes as loading control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!