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gst fusion protein


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#1 soymilk14

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Posted 25 September 2010 - 11:47 PM

hello everybody. I just induced protein of my gst-fusion (~32kda) together with gst control. Only gst was induced at 30 degrees for 4, 6, 8 hours. I don't see any induction in my gst-fusion after coomassie blue staining. But when i tried to purify it with gst beads, i saw 4 bands in my gst-fusion sample and the upper band was almost 32 kda.

My question is: can i do gst pulldown even if i dont see induction from 1 ml of broth i took for coomassie blue staining? Can my gst-fusion be not strong enough to be detected in coomassie blue?

any of your advise will be helpful... thanks so much...

#2 soymilk14

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Posted 25 September 2010 - 11:53 PM

i am attaching the picture for reference... the lane 1 and lane 2 from figure 1 are without induction. lane 3 is the gst control. lane 4 is the bacteria with gst fusion plasmid. gst-fusion protein not induced in lane 4.

However, when i did gst purification (figure 2) , i see 4 bands in my gst-fusion sample. lane 1 is marker, lane 2 is gst purified, lane 3 is the gst-fusion bacterial sample.

grateful for your advise.

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#3 ScienceRules

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Posted 27 September 2010 - 04:51 PM

I would be hesistant to do a pulldown with the information you have given. You should definitely be able to see your induced protein with Coomassie staining as you do so plainly with your GST control. I would consider doing a western blot and probing your lysate with a GST antibody to look for a protein of the right size.

Also, even though you have something that appears to be the right size bound to your beads, you also have several possible breakdown products, which is not optimal. Ideally you don't want any breakdown products. Either way your protein should definitely be in excess of the breakdown products. Are you using protease inhibitors?

#4 soymilk14

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Posted 28 September 2010 - 04:46 PM

thank you so much. i will consider your comments, and i am now trying to titrate the IPTG concentration, select several colonies and lower the temperature of induction. are these approaches correct?

yes, i used protease inhibitors to lyse and sonicate my bacterial pellets. i am grateful for your answer and for spending time taking a look at this. i hope i get better induction soon....

if there are any advise, tips or comments you may suggest, i will be very happy to learn from you all. thank you so much....

Edited by soymilk14, 28 September 2010 - 04:48 PM.





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