Hi all,
I am doing cell free protein reactions with His-tag (50 uL), and trying to purify it using Ni beads method (for small scale). After I finish purification and run western blot, there is no band showing up. But I got band before purification but weaker.
The issue I could think about: 1) since the protocol needs dilute samples with Equilibrium buffer in a 1:1 volume ratio, dilute more produced proteins, which is not detectable on western blot. 2) protein yield is not that high, and some of it in flow through, some of it in wash, and some of it in elution, should concentrate all samples to do western blot?
Is there any better way to solve the issue? or any better ideas?
Thanks!
Dear Rui,
I propose always check the presence of the recombinant protein before the purification (dot blot with anti-HisTag antibody, for example), because sometimes the efficient of its production is very low or it was not produced in this production trial. The second way is to scale-up the volume of the cell-free reaction.
I think that if you do not need protein sample with high purity level, you can pool all these protein fractions.
Good luck!
Chris
Agree with previous answer.
Depending on the size of your protein, I recommend spin concentrating the protein using a microcentrifuge concentrator. Likewise, it might be worth scaling up your reactions.
Can you estimate the amount of protein you are making by A280 or a bradford assay?
Kariona Grabińska
When I did western blot, I have not encountered issue or noises with other histidine in my cell-free reactions (I only get one band of my target protein with correct size). However, I found my protein is very sticky to Ni-beads, and cannot be eluted off, even by 500 mM imidazole.- Badges
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