I already extracted my protein directly in SDS buffer containing Bromophenol blue and also B-mercaptoethanol. After running SDS page, I am having problem with protein concentration. Is there any way by which i can concentrate my proteins in the buffer like we use vaccum centrifuge for DNA? I have more than 100ul of sample volume...please suggest
I suggest to put your protein in a dialysis tube (small diameter) with the pore size selected according to your requirements. Then lay this dialysis membrane in high-molecular dextran to remove the solvent in the tube by osmolarity. Afterwards you can shorten the tube with a third clamp very closely to the residual liquid in the tube. Then throw this tube in 1 to 2 l of your required buffer and dialyse it overnight. The advantage of this method is that you can prevent rigorous conditions. If there is a precipitate after concentration dont worry in most of the cases it is gone after final dialysis. I hope this helps to solve your problem. Kind regards
Protein precipitates out by acetone or any other organic solvent. So you can use that but I am not sure if any of the component of the lamelli buffer (SDS or B-ME) might interfere in this process.
I would not try to heat it for long time to reduce the volume and increase the concentration. Or may be heating at lower temperature, around 65°C might help evaporate the water but than u will not be able to find out the protein concentration.
@Omkar
thanks for replying,
I am also afraid of loosing my protein... already it is less in the buffer, i think.
how about using evaporation without using heat.is that compatible?
If the protein concentration is very low , you can heat the sample again at 99 degree. Keep the flap open, allow the sample to dry till 25-30 micro liter. estimate the protein again before running the gel. Second time heating usually do not affect sample quality. And there is no way of loosing your protein. I have done the same for many times. hopefully it will work for you too....
Ideally one should concentrate before adding to SDS buffer - however TCA-acetone precipitation should work - vacuum concentration at low temperature in a SpeedVac can also be tried.
I am little skeptical about heating though.
I suggest to put your protein in a dialysis tube (small diameter) with the pore size selected according to your requirements. Then lay this dialysis membrane in high-molecular dextran to remove the solvent in the tube by osmolarity. Afterwards you can shorten the tube with a third clamp very closely to the residual liquid in the tube. Then throw this tube in 1 to 2 l of your required buffer and dialyse it overnight. The advantage of this method is that you can prevent rigorous conditions. If there is a precipitate after concentration dont worry in most of the cases it is gone after final dialysis. I hope this helps to solve your problem. Kind regards
Protein concentrator tubes is the best option, Sorry I forgot to mention it. I just used them few days back and it is very fast, easy and convenient. be careful in selecting the pore size of the filter so you dont loose your protein. This method concentrates protein and at the same time also maintains the osmolarity of the solution as along with water (solvent), low molecular solutes (salts, detergents and reducing agents) will also be removed and so the osmolarity is maintained.
I have a question of the response of Mr. Dipankar Ash, how can we measure the protein concentration with protein in the lamelli buffer?
Dear Omkar, I think we can use the below mention protein estimation kit.
http://www.gbiosciences.com/ResearchProducts/CBX-desc.aspx#
Usually Lamelli buffer has bromophenol blue added into it so my question was how do you measure protein concentration when ur protein is already lamelli buffer. THe list of chemicals that does not affect the kit u mentioned does not include bromophenol blue or xylene cyanol. Thank you for your reply, this was helpful. I dint know companies make such kits also.
thank you all for your kind reply.
@ Dipankar Ash, dipjyoti - sir I did speed vaccum at low heat to concenterate my protein but i observed that protein concentration reduced and i could not detect my desired protein by immunoblotting.
@Jochen- thank you for your reply. your method sounds interesting. i never heard that before. i will search more about this technique and will try. thanks
about Protein concentrators which pore size works best. mine proteins are from 50kDa to 100 kDa size. This method sounds easy and convenient and also kind of fast.
thanks
Kind Regards,
Pratima
Although it is not mentioned, but the cleaning solution provided with the kit is able to clean bromophenol blue also. Accuracy can be checked with standard BSA solution.
Protein conc can be determined using 2D quant kit (GE Healthcare). We regularly use it for quantifying proteins in SDS loading or lysis buffer
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