Getting my primary antibody for Western - (Jan/29/2006 )
I am new to westerns. I have some 3 yr old transgenic trees and I want to look at the protein level from the gene of interest. This gene/protein will not be tagged in any way. After I isolate total protein, i'll run a SDS-Page, then I'll need my primary antibody to probe the blot with. How does this happen?
Can I make it from cDNA seq? Is there a company that people use that can make my primary anti-body for me from my cDNA seq? I am looking for speed (quality) over cost.
Thank you much!
If the protein that you are looking at is a common one than there are a plethora of companies out there. for eg cell signaling, neomarkers, santa cruz etc...
if the protein you are looking at is common, you should fine the company selling the antibody on biocompare
if not, you should contact a company to make the Ab. they will need the purified protein I would say? (to immunize mice or rabbits)
The protein I am after is not a common one and I'll need to purify it myself (I think). Can anyone give some info, link, ref., on doing this - I do have the cDNA seq. Can I some how pull it out with this?
if you are say the only lab working on this protien, and no one else has ever published a western etc then you have to have the antibody made... some universities offer this service, many companies do... search custom antibody synthesis on google...
before you go to all that trouble and cost you should really check the literature and companies, there are antibodies available commercially for alot of protiens, and if someone else used one for publication they usually have to provide it upon request even if it is not commercially available...
hope this helps...
So it sounds like its pretty complex (Ion exchange Chromo.) in getting from:
I see a phenotype in my transgenic plants therefore I know this plant has the protein TO being able to purify and quantitate that protein if: I know nothing about its protein:protein binding properties and if its not a fusion protein.
What about taking my gene, cloning it into a fusion construct, getting fusion protein, making my capture antibody from that and then using that to pull the protein out of my transgenic plants? This seems to be the best way I think. Please give me your opinion.
yes good idea.
make a fusion protein to purify it.
try to make so you can cut the tag, to avoid to synthetize some anti-tag antibodies.