I want to express and purify a protein of bacteria (approximately 121 kDa) using pGEX 4T-1, E. coli BL 21 and Glutathione Sepharose 4B. I have tried out with 0.1, 0.5 mM IPTG, at 30 deg C for 4 hrs. However, I do not see any fusion protein expression on the SDS-PAGE gel. Can anyone help me troubleshoot this? I would like to attach my protocol (Screening pGEX recombinants for tagged protein expression) and results of SDS-PAGE gel in the attached file.
Your pro expressed but your problem is, you loos your recombination pro at first wash.
Check your Sepharose.
Dear Ann
As Alireza suggest
from you gel, yuor protein seem to be expressed (band is present in the fraction 2 and not in the fraction 1). However in the past i experienced some cases where clones that do not express my protein show an induction band around 110KDa, that mass spectrometry identify as a contaminant but it was quite weak respect than your band.
However i tihnk that if you can do a WB with anti GST to be understand if this band is it your protein that do not bind the coloumn (is it aggregate?) or is a contaminant that casually run in the same MW of your protein.
good luck
Manuele
Hi An,
First of all I would recommend you to check if your protein of interest is expressed at all. Do a western blot with anti-GST antibody and check IPTG controls, suspension, supernatant and pellet with that. To be safe you can check your wash and elution fractions too. If you obtain a band at about 150 kDa your protein is expressed but probably cut through protease. If not, you have a problem with the expression. You have definitley expressed GST. I can highly recommend the GST-resins from Cube-Biotech. I always got a high protein yield with them (https://cube-biotech.com/products/purification-resins/gst-affinity-resins/#).
- Badges
- Science topic
- Similar topics
- Filing