knuf

Frequently 3 are required, but I've never seen more than 3.

Yes, phosphorylation can cause your apparent molecular weight to be off--as well as other post-translational modifications. One thing that is often forgotten in SDS page is that it separates based on both size AND CHARGE, and while it is generally assumed that SDS coats proteins equally, thus giving each the same negative charge, this isn't always the case (see work with Tau protein, which is known to run at a different apparent molecular weight than its amino acid composition would suggest). Also, if the protein is not completely reduced, remaining dilsulfide bonds can cause the protein to run aberrantly by altering the molecular size of the protein (which incorporates both weight and length). Finally the lipophilicity of the protein can affect the apparent weight, which i guess is due to the charge similar to SDS coating--see work with LC3 in the autophagy field--when the protein is conjugated to a lipid molecule, although the actual molecular weight is higher, it runs smaller due to the added lipophilicity.

Hi! I am wanting to know if anyone has had success propagating and creating competant ccdB survival cells from Invitrogen. These are the ones necessary for propagating destination vectors for gateway cloning. I realize you can just buy them (which we have and thus, have a stock that we can propagate), but considering how expensive it is to buy them, and how cheap it is to grow bacteria, it seems silly to continue to buy them. So have people done this? Are there any special considerations for these cells versus other cells?