ha-tag purification , lots of bands - (Dec/11/2006 )
hello, i am doing protein purification with HA affinity matrix from covance. i made a construct to express my protein and with HAx3. then i did transfection, with ha-antibody , i can see that the transfection is successful . i can see a specific band of my protein with ha (43kd) when i do western. but the problem is after i use the ha affinity matrix to purify it and use ha peptide to elute the protein, i got lots of bands on the silver staining. i follow the instuctions of covance, so it supposed to show a clear band at 43kd after i use peptide to elute it.
has anybody did this before?
basically you can increase the salt concentration of your incubation buffer. Will reduce the non specific.
Other possibility is to repurify the purified fraction. Should reduce the background but not perfect.
i saw some graph from the companies that sell the ha affinity matrix. for example roche shows a good graph of the purification, they got a single band after elution. i do the exact thing and use the buffer they used. i don't know why i am not even close to what they get.
we found that for some protiens, affinity purification always gives some background. U could try playing with the salt concentration. But as fred suggested try to repurify, u may not completely get rid of the other bands.
probably these bands are the protein's binding partners.