I've got a protein gel mystery.
I took exactly the same frozen cell lysate and resuspended it in two different buffers and the western blot looks completely different.
In PBS +triton, I have a crisp band of the protein of interest at the top (green), a known cross-reacting band, and a small band (yellow ??), in HEK5G+chaps the band of interest is blurry and the small band is missing.
What is happening here?
PSA for #westernblots
Did you know you can often leave out the methanol and SDS and get an excellent transfer?
Our recipe for 4 liters of 10 x is 560g Glycine, 120g Tris Base.
We transfer at 0.5mA for 30 min for most proteins 40 for >150kDa in a biorad rig.
We use an ice block but no stir bar. It gets hot so don't go longer than 40 min.
Does anyone else think we need hashtags for when you need help with your science?
How about
#MolBioHelp
#ProteinScienceHelp
#ImmunofluorescenceHelp
#WesternBlotHelp
MCDuncanLab