I was successful with the transformation( pPIC9K vector in Pichia pastoris GS115 strain )and got good colonies in RDB plates after 48 hours. later in MD and MM plates also. Can anyone please tell me how to check the expression of those colonies before going to large scale expression.
Just do a small scale expression in 5-10mL.
I never tried that, but if you have a constitutive promoter you could maybe just resuspend your colonies in a small amout am Lämmli.
Do you have any tag attached to your protein? The best would be to prepare a small liquid culture, pellet the cells and perform an SDS-PAGE or Western Blot to be sure that your protein is expressed. Be careful with protein concentrations that you depose on each well in order to have some nice results.
My protein dont have any Tag . I just want to screen almost 50 colonies to check which one gives best expression and use them further for large scale expression
@Jorgaq Pata
Vinny Vinod and you have to sceen all of them? I mean, you can screen some colonies to check whether the expression varies a lot or no if your colonies are the same in theory. But you have to do small volume cultures in order to get some material, lyse the yeast cells and check the expression since you don't have any tag that could help you a bit (ex.GFP)
Thank you so much guys Jorgaq Pata & Simon Flueckiger I will try and see if its a success I will definetly share the results :)
Vinny Vinod just be careful with the lysis, it is difficult to properly lyse yeast cells, you can check them on microscope to see whether they are lysed or not.
Jorgaq Pata I am planning to add lyticase if I am lysing but if you have a protocol do share with me.
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