I want to perform a transference of proteins from a gel fixed with acetic acid:ethanol:H2O (no coommassie) to a nitrocelulose membrane using a transblot SD (semi-dry, Bio Rad). Does fixing affects the transference?
Fixing denatures protein so that it is immobilized, which is what "fixed" means. Clearly your fixed gel will not transfer the way an unfixed gel would. Also, in acetic acid most of the proteins are positively charged (protonated) and will run in the opposite direction of what is required for normal transfer. The fixed proteins also have most of the SDS stripped off them, which would otherwise have given them the large negative charge required for normal transfer. However, you may be able to "rescue" your gel by soaking it in a mild alkaline buffer to neutralize and remove the acetic acid (several hours with agitation (check the buffer pH and replace if necessary)) and then in running gel buffer containing SDS (also several hours with agitation and maybe a buffer replacement). After that preparation a lot of proteins will transfer normally (with some losses and band broadening), but some will not. It depends on your protein. It's worth a try if you are desperate, but why fix in the first place? Just run another gel and transfer it without fixing. Anyway, good luck.
Fixing denatures protein so that it is immobilized, which is what "fixed" means. Clearly your fixed gel will not transfer the way an unfixed gel would. Also, in acetic acid most of the proteins are positively charged (protonated) and will run in the opposite direction of what is required for normal transfer. The fixed proteins also have most of the SDS stripped off them, which would otherwise have given them the large negative charge required for normal transfer. However, you may be able to "rescue" your gel by soaking it in a mild alkaline buffer to neutralize and remove the acetic acid (several hours with agitation (check the buffer pH and replace if necessary)) and then in running gel buffer containing SDS (also several hours with agitation and maybe a buffer replacement). After that preparation a lot of proteins will transfer normally (with some losses and band broadening), but some will not. It depends on your protein. It's worth a try if you are desperate, but why fix in the first place? Just run another gel and transfer it without fixing. Anyway, good luck.
Thanks a lot for your answer Norbert. It was just an experiment to kwown if I could store a gel (fixed) for a few days and then transfer it to a membrane. But you are right. When I made the question I was already running a new gel for making a typical transference. thanks again!
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