Nov 12, 2023
Immunoprecipitations and Western blots analysis
- 1University of California, San Diego
Protocol Citation: Leonardo A Parra-Rivas 2023. Immunoprecipitations and Western blots analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3ep5lk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 12, 2023
Last Modified: November 12, 2023
Protocol Integer ID: 90818
Abstract
Immunoprecipitations and Western blots analysis
Immunoprecipitations were performed using
1-2 mg of total protein. Samples were incubated overnight with the indicated
antibody at 4° C, followed by the addition of 50 μl of protein G-agarose beads
(Thermo Scientific, Cat#20397).
Immunoprecipitated proteins were recovered by centrifugation at 2,500× rpm for 2 min, washed three times with a buffer containing PBS and 0.15% Triton X-100. The resulting pellets were resuspended in 20 μl of 1X NuPAGE LDS sample buffer (Thermo Scientific #NP007) and
incubated at 95 °C for 10 min. Samples were separated by NuPAGE 4 to 12%
Bis-Tris polyacrylamide gels (Thermo Scientific #NP0335BOX), and transferred to
a 0.2 µM PVDF membrane (Thermo Scientific #LC2002), using the Mini Blot Module
system (Thermo Scientific).
PVDF membranes were first fixed with 0.2% PFA 1x PBS per 30 min at room temperature. Then, membranes were washed three times for 10 min in PBS with 0.1% Tween 20 Detergent (TBST) and blocked for 1 h in TBST buffer containing 5% dry milk, and then incubated with the indicated primary
antibody for 1 h in blocking solution, washed three times for 10 min each and
incubated with HRP-conjugated secondary antibodies (RRID:AB_2819160),
(RRID:AB_2755049).
After antibody incubations, membranes were again washed three times with TTBS buffer, and protein bands were visualized using the ChemiDoc Imaging System (BioRad) and quantified with Image Lab software version 6.1 from BioRad (RRID:SCR_014210)
http://www.bio-rad.com/en-us/sku/1709690-image-lab-software.