I have extracted proteins from rat heart tissues and performed a western blot analysis on the same. Used cmyc antibody and cfos antibody purchased from Santa Cruz Biotechnology. Bands are detected but not at the proper band size. The control- Actin shows up properly in every single experiment performed.
Use either Sigma or Cell Signaling Technology antibodies, Santa Cruz failes all the time. They are not whatt the brand implies. I have used both brands repeatedly, CST antibodies are best signalling related proteins and for their phospho products which I assume you may want to show since you are working with transcription factors (c-myc/ c-fos). Sigma antibodies works fine overall.
CST' s abs are really good. Try to reduce your extracts more with more sds, beta-mercaptoethanol or dtt to avoid dimerization of your proteins that can change the mw in the wb.
If the protein is detected at higher mol wt especially in multiple of original Mol wt then increasing the denaturing conditions as suggested by Jose might help. Also try to use lysate of cell lines (HeLa) and see if you get them at right size (sometime working with tissue are problematic for proper lysis and denaturation). If not just change to a better antibody.
maybe you should try to fine any information from www.uniprot.org.,.,.,,., the site have alot of information about protein “The mission of UniProt is to provide the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequence and functional information.”
Thanks guys...will try adjusting my protocols accordingly...
@ Abhishek ...I have used dilutions from 1:200 to 1:5000.....all of my blots have given the same result....bands observed at the incorrect band size..
I tend to agree with Kanikkai Raja. Your proteins are possibly getting degraded during extraction. You should use a cocktail of protease and phosphatase inhibitors available commercially to minimize protein degradation during extraction.
Hi Sagarika,
Best control for the antibody will be the postive control that company showed in the product sheet. As they claim it works. In you situation, I would call the company and let them know about the band you are getting. Santa Cruz antibodies are okay but they have a good coustomer service, In past they have provided me with free positive control and/or new vial of the antibody.
Also if molecular weight of you protein on the blots can be altered (only few KDs) with post translational modifications (e.g. phosphorylation etc.) also not useing correct % of the gel and denaturing conditions can effect migation of the proteins also.
Good Luck.
hardik
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