I am trying to isolate a recombinant protein GST-GSK3b for my dissertation research using FPLC (fast protein liquid-chromatography). The protein is expressed in Sf9 cells and infected with baculovirus. The cultures have been incubated at 27 oC for ~72h post-infection and the % cell viability at time of harvest was > 60%. I am using a 1mL HP GSTrap column on the FPLC to isolate this protein.
When I am doing the FPLC experiments, I analyze a small amount of the lysate input in the FPLC, and a small amount of the FPLC flow-through lysate using 40% GST slurry. The buffer conditions used are 50mM TRIS-HCl pH 8.0, 50mM NaCl, 1mM EDTA, 1mM DTT and 0.1% (w/v) Triton x-100.
As shown in the SDS-PAGE images, the recombinant protein (~73kDa band indicated with the red Asterix) is definitely expressed in the culture, and it has bound to the glutathione beads when incubated with 40% GST slurry. But a significant amount of the recombinant protein is present in the FPLC flowthrough and no recombinant protein is detected in the FPLC elution fractions. The elution buffer contains 10mM GSH.
Why is it this protein is able to bind to glutathione beads in the GST-mini pulldown experiment but not binding to the glutathione beads in the column? The buffer conditions used for the GST-mini pulldown experiments and the FPLC experiment are the same.
For the GST-mini pulldown experiment, I incubate the lysates with the beads at 4 0C for 2h. In the FPLC, I pass the lysate through the column at 0.3mL/min, so if I pass ~10mL of lysate through the column it takes about 33-35min.
Can anyone share a possible explanation for this GST tagged protein binding to beads in the slurry but not the beads in the column in the FPLC? I would appreciate any thoughts, input and feedback. Thank you!
Hi,
What's the temperature of the other setup? If you are using the same amount of beads in both setups that leaves you with either time or temperature. Either increase the time of flow (if that's possible) or increase the amount of the beads? Temperature is also worth checking.
Hi Bhagya,
Couple of thoughts:
a. I second with Leonid, does your setup allow you to further reduce the flowrate? or can you try a 5ml column? I am assuming the temp maintained around the workstation is 4-8degC.
b. Can you try reducing the Triton (10 or 50 times) in your buffer, for FPLC run.
c. Did you check the over-expression in the crude cell-sup, before involving into any levels of GST-pulldown? This can help you with estimating the proportion of exogenous levels and you could later decide to concentrate the lysate too.
d. Also, as an extreme, can you Western Blot your mini-IPs and confirm what you are seeing in CBB is indeed your protein of interest?
Hope it helps!!!
Leonid Ouchanov Thank you for your response Sir. The FPLC instrument is at room temp. I usually keep my FPLC buffer continuously incubated in an ice bucket to keep the buffer flowing through the system cold, but the instrument itself is at room temp. (~23-25 0C). I will try reducing the flow rate as you suggested.
P. Vineeth Daniel Thank you for your comments and feedback. Yes, our set-up allows to further decrease the flow-rate. Thank you, I think this would be a good thing to try. The reason we started to include 0.1% w/v triton x-100 in our buffers was because it improved protein elution from glutathione beads at the GST-mini pulldown level. Before, adding triton x-100, a lot of the protein still remained bound to the beads, even after attempting elution with 10mM GSH for several times. But, you are right, perhaps the triton x-100 could be having some effect on the binding? We have previously confirmed that the 73kDa band we see is indeed our recombinant protein. In your comment (C) are you asking if we have done like a western blot for the crude lysate? If so, no we haven't done that without trying the GST-pulldown.
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