I have expressed my protein in pichia using a secretory signal and it is having two tags His and Myc,
After induction I performed ammonium sulfate precipitation of protein from supernantant (600ml) to concentrate, (50% saturation)
I checked expression of protein using antimyc antibody and clear band was visible.
Then I dialyzed the conc. protein and performed Ni-NTA purification using 2 ml of each of different con. of imidazole (50mM, 70, 100, 150, 200 and 250),
Before elution I incubated my conc .protein solution with Ni-NTA agarose beads(Qiagen) for overnight.
However after comissie blue staining of SDS-gel of these eluted fractions I didn't see any band corresponding to desired size (22.5kda),
3-4 bands of larger size proteins were visible (150-72 kda) in all eluted fraction esp. after conc. of 100 mM imidazole onwards
In the flow through also, my protein was not detected however many of other proteins were visible
I think either the protein amount is very less so I should grow more volume of culture
Apart from it, Kindly help me to troubleshoot this problem.
Dear Mamta
I can comment on the gel:
Comassie staining is not that sensitive, have you tried Silver staining?
Also, in general these methods bind mostly to specific aminoacids.
Here a summary that I found: https://www.thermofisher.com/de/de/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/protein-gel-stains.html
Have you tried to visualize your protein via westernblot after all your procedure?, you could evaluate the protein in every step in order to see in which step gets lost and optimize that.
It could also be that what you observe is your bead with your tag protein and without any of your protein (just guessing)
Regarding the use of of NiNTa I found this, might be of your interest:
https://www.researchgate.net/post/Protocol_for_Histag_based_Ni-NTA_protein_purification
Dear Nelly
Thanks for your feedback. I haven't done silver staining or western blotting after all the procedure. I need threprotein atleast in microgram quantity. after reading the protocol in link u mentioned, I think I need to change some of the steps in my Ni-NTA purification procedure.
In this case, I can give several suggestion:
a. Try to grow enough culture (100 mL of culture is enough if you still optimize the condition)
b. Try to use low concentration of imidazole during washing process and increase your imidazole concentration in final step (Elution step).
c. Collect 10 - 20 ul of sample in each step (lysate, flow through, eluate 1 and eluate 2) for checking your protein concentration in western blot.
d. In western blot, you can see in what steps your protein concentration decrease as you can see from the bands
e. If you still face the same problem, try to use another beads such as Talone beads. Or you can disrupt your cell with another methods such as glass beads.
Good luck!
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