Hi, I tried to express membrane proteins in a cell-free system with the addition of liposomes, when i take a sample of the reaction and mix it with SDS loading buffer and incubate at 37C for 1 hour, I am seeing no band, the control is visible (not a membrane protein and no liposomes).
My question is for the liposome inserted membrane proteins, is just incubating in the SDS loading buffer at 37C enough to release the proteins from the liposomes?
Is there something else i can do to see my protein band?
There is probably not enough sds to dissolve the liposome and denature the protein. I would add tween or triton or something like that to first disrupt the liposome.
I have refolded proteins using liposomes and inserted proteins into liposomes and then analyzed these by SDS-PAGE and had no problem. However, these were NOT membrane proteins.
The sample buffer with SDS and DTT, as well as the heating should be enough to disrupt the liposome and allow your protein to enter the gel freely.
I some suggestions: use a control that has your membrane protein but no liposomes in a protein gel to see if the protein is soluble enough and not large enough sot that it can migrate in your protein gel system. If the protein cannot enter the gel, then you need to consider solubilizing the protein in extra detergent in order to keep it from precipitating in the well and entering the gel. When you stain the gel, check to see if there are any high proteins just below the well- this will indicate precipitated proteins- usually membrane proteins.
Hope this helps.
Dear Gustavo,
I do not have the membrane proteins, i guess i phrased it wrong, i didn't insert them into liposomes, i expressed them using liposomes and theoretically if they got expressed they got inserted into liposomes. So i cannot have a control without liposomes.
Also there is nothing under the well after staining.
As you and Omar suggested, i will try tween ad triton and see the result. It might also be a concentration and detection sensitivity issue, as i am using coomassie blue , but theoretically the company specified "From our experience the protein yield is range from 200ng/uL reaction to 1000ng/uL reaction" so i should have minimum 1000ng expressed in the 5ul sample i am loading, and coomassie should be able to easily stain as little as 50ng.
So i think i should not waste sample and load 10ul instead of 5ul i don't think that would make any difference. Expression either failed, or there is a solubility issue as you are suggesting because non-liposome DHFR was clearly expressed as a control.
Thank you for your suggestions.
Based on your feedback, it sounds like an expression issue. I really do not think that the triton/tween will help much. You need more SDS since it wraps around your protein better- making it more negative and polar so it will enter the gel.
Gustavo , I agree.
I'm now suspecting residual RNAse from the miniprep kit is maybe causing issues ... I am considering doing miniprep without the RNAse since getting my hands on RNAse inhibitor will take months... Maybe run a gel with the contaminating RNA+plasmid purify the plasmid after miniprep? Do you think that's a good idea?
I would think that is the next step. I hope you can get your hands on RNase soon since it would be better to run the protocol as it should be.
Hi Vicken,
So if you expressing proteins (hydrophilic) using cell free system in liposome, why you think protein should be inserted into liposomes? The membrane/liposome insertion of hydrophobic protein is thermodynamically favourable and hydrophilic protein specially with charged residues likely not to go into liposomes. Thats how I thought.
However, I have successfully run SDS-PAGE of liposome inserted membrane proteins by simply solubilising the liposomes with triton or Fos choline 12 for 30min followed centerifution to pellet the insoluble fraction (which is usually less than 5% of the sample if you use harsh detergents such as FC12. I then run SDS-PAGE with SDS sample buffer. I DO NOT recommend heating unless you know it works, as it may cause protein aggregation and larger aggregate may not even enter the gel.
Good luck!
Shahid I am expressing membrane proteins , so it is likely they favor insertion into liposomes, but inserted or not , either way i want to see expression i do not care about the insertion. I assume membrane proteins would be expressed poorly and aggregate without liposomes or nanodiscs or something of the sort.
I tried with triton , no change, nothing expressed. so I'm going to try purifying my plasmid from any RNAse as i said above.
Thanks :)
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