separating protein and nucleic acids - problem separating protien and nucleic acids (Aug/08/2008 )
Hi,
We were doing protein extraction and estimation from a DH5α after inducing protein expression using IPTG. 10ml of the broth culture (pellet) was washed using 1ml 10mM Tris- HCL pH 7.4 twice and spun to pellets at 14000rpm for 2 mins. pellet was resuspended in 0.5ml cell lysate buffer and was subjected to heating for 3mins (101C). It was centrifuged at 14000rpm at 5mins. The suspension was transferred to a new eppendorf. This produced a uniform layer of viscous solution which had nucleic acids and proteins. We didn have any DNase or RNase for degrading the nucleic acids. Rather it was sheared by syringe method/ applying pressure, still we didn get any separation. How will i separate protein and nucleic acids and how to reduce the viscosity. The final protein concentration was found to be 2ug/1ul(lowry's method). What could i have done to improve the protein concentration?
An interesting protocol.
Are you sure the proteins will survive the heat treatment? Why did you choose this questionable protocol to begin with? What’s your objective, to get proteins or to isolate DNA?
There is no mechanism included in the protocol that separates proteins from DNA. All you did is that you shredded DNA into smaller fragments by passing through a needle. Now you got a mix soup of protein and DNA fragments.
Please help us to better understand your objectives so we can better help you. ;-))
I agree with genehunter-1 -- 3 minutes at 101 C is pretty harsh for a protein isolation. Can you get some DNAse and RNAse? This would be the easiest way to get rid of nucleic acids, but of course adds proteins to your mixture. Do you have a sonicator or a French press available? You could also precipitate the proteins from solution using ammonium sulfate; I think the nucleic acids would remain solubilized, but someone else may jump in here if I'm wrong about that.
Hello Genehunter1,
Thanks for the reply. To begin with no we were not provided with anything else. This is a crude method, and i am sure that heating would unwind the proein structure but the time period for heating the mixture is too short to degrade a protein. This being DH5a and the protein i am looking for is b galactosidase i think it can withstand the heat for 3 mins. But anyhow there was no separtion of protein and nucleic acids, and no we were not provided with any DNase or RNase.
hello homebrew,
We were not provided with anyother things, like a french press or sonicator or chemicals like DNase or RNase. Also i believe b-galactosidase the protein i am looking for is heatstable for 3 mins. The heating was mainly employed to induce the reaction for cell lysis. Thank for the tip on ammonium sulphate. Will try doing that.
As far as I know HomeBrew is right - ammonium sulphate would precipitate proteins and leave nucleic acid in solution.
I hope you are working with some heat resistant form of b-Gal. :-))
maybe you can try basic polymers such as PEI as DNA precipitation agents. some ppl use cationic detergents such as CTAB as well. both work with the same principle.
Hi
this is really stupid to isolate the proetin by just heating the cells.But if u want that protein in denatured form try to to run a gel and cut the gel peace(protein band) and try to extract your protein (for raising Abs). but isolating the pure protien fraction by just lysing the cells is not possible.Because the no of fragments you are generating at 100C is very high(i dont think your protein is stable in confirmation at that temp). so try to take more amount cells and try to develop a lysis method( can be lysozyme or french press or glass beads or ......) and try to develop some separation technique to get pur fraction.
Pure fraciton is always best rite
Reagrds
S
We were doing protein extraction and estimation from a DH5α after inducing protein expression using IPTG. 10ml of the broth culture (pellet) was washed using 1ml 10mM Tris- HCL pH 7.4 twice and spun to pellets at 14000rpm for 2 mins. pellet was resuspended in 0.5ml cell lysate buffer and was subjected to heating for 3mins (101C). It was centrifuged at 14000rpm at 5mins. The suspension was transferred to a new eppendorf. This produced a uniform layer of viscous solution which had nucleic acids and proteins. We didn have any DNase or RNase for degrading the nucleic acids. Rather it was sheared by syringe method/ applying pressure, still we didn get any separation. How will i separate protein and nucleic acids and how to reduce the viscosity. The final protein concentration was found to be 2ug/1ul(lowry's method). What could i have done to improve the protein concentration?
hi shan,
Who talked about purifying the protein, we were trying to islolate the protein in a cost effective manner, now that we know the effect of other factors, we will proceed cautiously. Also i do understand the denaturing properties of heat on proteins, but we are not really bothered abt getting the exact chemical structure. So chill out, anyway thanks a million for the reply, i appriciate it.
Regards
Miraclestrain