Urea-soluble protein extracts from cells and tissues were prepared as described (38 (link)). Proteins were size-fractionated on 4–12% gradient polyacrylamide Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes. The membranes were blocked with Odyssey Blocking solution (LI-COR Bioscience, Lincoln, NE) for 1 h at RT and incubated with primary antibodies at 4° C overnight. After washing the membranes with PBS containing 0.2% Tween-20, they were incubated with infrared dye (IR)–labeled secondary antibodies at RT for 1 h. The IR signals were quantified with an Odyssey infrared scanner (LI-COR Biosciences). The antibodies and concentrations are listed in Table S1 .
Gradient polyacrylamide bis tris gels
Manufactured by Thermo Fisher Scientific
Gradient polyacrylamide Bis-Tris gels are laboratory equipment used for protein separation and analysis. These gels provide a gradient of polyacrylamide concentration, allowing for the separation of proteins based on their molecular weight. The Bis-Tris buffer system maintains a neutral pH during electrophoresis.
Automatically generated - may contain errors
2 protocols using gradient polyacrylamide bis tris gels
1
Urea-Soluble Protein Extraction and Western Blot
2
Investigating B Cell Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly isolated splenocytes were enriched for B cells using CD43 magnetic bead–based depletion and stimulated in RPMI 1640 with or without stimuli. Cells were collected at various time points and lysed with 1% SDS. Lysates were run on 4–12% gradient polyacrylamide Bis-Tris gels (Invitrogen). The resolved proteins were transferred to polyvinylidene difluoride membranes using the BOLT transfer system (Thermo Fisher Scientific). The following antibodies were used to probe for various proteins: Regnase-1 (GeneTex), Bcl2 (BD Biosciences), Vinculin (Cell Signaling Technology), and b-Actin (Cell Signaling Technology).
For the immunoblots with GC B cells and follicular B cells (Fig. 6 B ), wild-type mice were immunized with SRBCs, and GC and follicular B cells isolated by magnetic bead–based depletion using the strategy previously described (Cato et al., 2011 (link)).
For the immunoblots with GC B cells and follicular B cells (
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!