BMMCs or MC/9 were stimulated as described above. Cells were lysed in 1% Nonidet-P-40 lysis buffer (20 mM Tris-HCl, pH 7.5, and 150 mM NaCl) supplemented with AEBSF, aprotinin, leupeptin, pepstatin, sodium fluoride, sodium orthovanadate, and β-glycerophosphate on ice for 20 min. Lysates were centrifuged at 4°C for 15 min. For Western blotting analysis, SDS-containing sample buffer was added to lysates before loading onto 10 or 12% SDS-PAGE gels when analyzing for FcεRI β and γ chains. For IPs, lysates were incubated overnight at 4°C with appropriate antibodies. Immune complexes were precipitated at 4°C for 2 h with protein A or G agarose beads (Thermo Fisher Scientific) for rabbit or mouse Abs, respectively, then washed with NP-40 lysis buffer three times before analysis by SDS-PAGE gels. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes using the PierceG2 fast blotter (Thermo Fisher Scientific), probed with appropriate antibodies, and imaged on a ProteinSimple Fluochem M cooled charge-coupled device imager (ProteinSimple).
Protein a or g agarose beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protein A or G agarose beads are a type of chromatography resin used for the purification of antibodies and other proteins that bind to Protein A or Protein G. These beads are made of agarose, a polysaccharide derived from seaweed, and are functionalized with either Protein A or Protein G, which are bacterial cell wall proteins known for their high affinity to the Fc region of antibodies. The beads can be used in a variety of chromatographic techniques, such as affinity chromatography, to selectively capture and purify antibodies or other target proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using protein a or g agarose beads
1
Immunoprecipitation and Western Blotting of FcεRI Signaling
2
RANKL-Induced RANK/TRAF6 Coimmunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The coimunoprecipitation assay were performed as previously described21 (link)22 (link). Briefly, the RAW264.7 cells were seed in a 6-cm dish and pretreat with indicated concentration of XN for 4 hours and then the cells were incubated with RANKL (30 ng/ml) for 20 minutes. The cells lysate supernatant were added with the RANK or TRAF6 antibody. After incubated with the protein A or G agarose beads (Thermo), the immunoprecipitated protein were subjected to Western blot using TRAF6 or RANK antibody.
3
Immunoprecipitation Assay for PlexinA1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoprecipitation assay, cells were harvested and lysed in radioimmuno- precipitation assay (RIPA) buffer. Cell lysates were then centrifuged at 12,000 rpm for 10 min at 4°C (Hayashi et al., 2012 (link)). PlexinA1 or IgG antibodies were added into the supernatant and incubated at 4°C overnight with rotation. Protein A or G agarose beads (Thermo, Rockford, IL, USA) were then added and incubated with rotation for 3 h at 4°C. After centrifugation, proteins were subjected to Western blotting.
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!