In all, 1 × 106 cells were grown in T25 cell culture flasks, when cells were 80% confluent, they were lysed using the CHAPS-lysis buffer (Cell Signalling #9852). Protein was collected by centrifuging the cell lysates at 12,000 x g. Protein homogenates were then incubated with primary antibody (1:200) overnight in cold room. Antibody-protein complexes were separated by Pure-Proteome magnetic A/G beads (Sigma LSKMAGAG02) using manufacturer’s protocol and run on an immunoblot to detect interacting partners.
Chaps lysis buffer
Manufactured by Cell Signaling Technology
CHAPS-lysis buffer is a reagent used for cell lysis and protein extraction. It contains the zwitterionic detergent CHAPS, which helps to solubilize and extract proteins from cells. The buffer is designed to maintain the native conformation and activity of proteins during the lysis process.
Automatically generated - may contain errors
Lab products found in correlation
Phosphor akt, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Eif2α, by Cell Signaling Technology (1 mentions)
Phosphor eif2α, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
Phosphor pi3k, by Cell Signaling Technology (1 mentions)
Smac diablo, by Cell Signaling Technology (1 mentions)
Phospho ire1α, by Cell Signaling Technology (1 mentions)
Ire1α, by Cell Signaling Technology (1 mentions)
2 protocols using chaps lysis buffer
1
Immunoprecipitation of Protein Complexes
2
Immunoblotting Analysis of Apoptosis and UPR Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested after treatment, washed 3 times with PBS and lysed in CHAPS lysis buffer (Cell Signaling Technology). The concentration of protein was measured using the BCA protein assay kit, and loading buffer was added to the samples. The samples were subjected to 12% SDS-PAGE; then proteins were transferred onto PVDF membranes (Millipore). After blocking with 5% non-fat milk at room temperature for 1 h, PVDF membranes were incubated with primary antibodies overnight at 4°C. The following antibodies against these proteins were used: Caspase-3, PARP, Mcl-1, Bcl-xl, Bcl-2, Bax, Smac/DIABLO, Cytochrome C, ATF-4, phospho-IRE1α, IRE1α, phosphor-eIF2α, eIF2α, phosphor-Akt, Akt, phosphor-PI3K, PI3K, mTOR, and GAPDH, which were all purchased from Cell Signaling Technologies (Danvers, USA). Next, the membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature.
Finally, the proteins were detected by enhanced chemiluminescence, and densitometric analysis was performed using ImageJ software. The cytosolic fraction of the cells was purified as described earlier [25] .
Finally, the proteins were detected by enhanced chemiluminescence, and densitometric analysis was performed using ImageJ software. The cytosolic fraction of the cells was purified as described earlier [25] .
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!