Polyclonal antibody production using SDS-PAGE gel fragment - (Dec/01/2011 )
I need to produce antibodies for CO-IP, but my protein of interest is produced as inclusion bodies and many expression optimisation / refolding attempts have failed. Do any of you know of a protocol that uses SDS-PAGE gel fragment directly for antibody production? I've seen that certain antibody production companies only require you to send them a dried SDS-PAGE band, but i cant manage to find instructions! Will the presence of SDS affect the immunization? Surely?
the sds present will have no significant effect on the immunization. keep in mind that you will be generating antibodies to denatured protein and may not recognize native protein.
Hola,to me the main requirement to extract protein of a gel is don´t fix stain because protein remains fix to acrylamide and it isn´t easy extract it. (I don´t know if people immunizes with an blue suspension of acrylamide). Some years before, we run preparative gels with a bit of Comassieblue in the upper buffer and after run we cut band, pass throught a 10-20 ml srynge and homogenize with a tisssue blade homogenizer extracting of the suspension some times untill the supernatant were less blue. after dyalisis and liophilization and reconstitution you will inject a blue solution to rabbits or mice. Buena suerte
we've injected fixed, stained and mashed up gel slices and had excellent results (we do wash out the methanol/acetic acid prior to homogenizing the gel slice in pbs).