Dear May,

For what purpose do you want to make pores in the cell wall? Indeed, Zymolyase could be a too rigorous treatment. I think that DTT or DMSO are a good options for you. These chemicals are mostly used for yeast transformation. One question: what is OG

Octyle glicoside- detergent-short-chain

i need to make pores for a ligand to enter

thank you

I would try to use some pre-oligomerized pore-forming toxin or peptide instead of OG.

The question is, would you like to make pores in the plasma membrane or loosen up the cell wall a bit, so that your ligand readily reaches the plasma membrane? Octyl glucoside will have no effect on the cell wall, neither a pore-forming peptide.

thanks for the answers!

i like that i will have pore in the cell wall but the mambrane will stay as she is. i have a mambrane protein and i wish my ligand will cross the cell wall but that the cell wall will still be there- so the ligand will enter through pores and then will be connected to the protein on the membrane

You are welcone :). I recommend you to test a range between 1 and 30 mM of DTT in TE buffer. Incubate your cells between 30 and 60 min at room temperature. Then wash your cells a couple of times. (do not centrifuge at high speed because your cells have a weaker cell wall now, i recommend 3000 g for 2 or 3 minutes). Good luck!

@albert de boer

i am trying it this week. you think i could use PBS buffer? why TE buffer?

thank you

i also want to try using LYTICASE do any one know how should i use it?

Hello May,

PBS is fine too. You probably have already tested it.

I have never used lyticase, but I guess it wouldn't be very different from using zymolyase? You have to look up the appropriate buffer. Did the DTT work?

Hey 

the LYTICASE/DTT all didnt work for my plan. 

but now i am looking for somthing alse i wish you can help me

i need  plasmid that will have gpcr+sequence that will lead him to the membrane of a yeast

up until now it was genomic one. i need it in a plasmid. 

maybe you know?

yjamls so much for your help!!!