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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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protein precipitation after IMAC
Posted by: aga (IP Hidden, Unregistered user, )
Date: March 4, 2005 05:25AM
Hi,
I'm working on 35kDa protein with HIs-tag N-terminal. After purification on Ni-column (Qiagen) I usually set up the dialysis o/n and the protein always precipitates. It doesn't matter if I use Tris, HEPES, CAPS. I tried pH 4-10. I add 20% glycerol, Bme, I try to elute using low pH instead of imidazole, I increased the NaCl concetration, also dilution of my eluted protein, I did the dialysis at 4C and also at room temp... nothing helps. Only what I can say - yes I;m sure I have my protein, it's really pure on the SDS gel and I get quite a lot of it after IMAC. Do you have any ideas what else I could try?? Thanks in advance for your help!!! Agata
Re: protein precipitation after IMAC
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: March 14, 2005 11:44AM
This is a quite common problem that purified proteins tend to precipitate if dialyized. However, to overcome such problem you need to exchange your dialysis buffer every 30 minutes for the first hour and half and then leave (3 times) and then extend to an hour (2 times) and then dialyze over night. This gradual dialysis approach will help in eliminating the ppt problem you are facing.
Good Luck!
Re: protein precipitation after IMAC
Posted by: zia (IP Hidden, Unregistered user, )
Date: March 15, 2005 09:34AM
respected sir,
i am working on very small amino acids sequencing protein (antifungal protein), but the problem is elution and purification of proteins. after centrifugation these proteins are present in supernatent and the next step is presipitation of proteins. what method is best either by salt or organic solvent. the problem is that the required protein is present in very small amount and in supernatent a large amount of different protein is present. what i do in this case? electrophoresis is good method for this or hplc. but the problem in hplc is that there is no specific methedology as of mobile phase or elution time and so on. so i think electrophoresis is best. comment please zia
Re: protein precipitation after IMAC
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: March 15, 2005 07:03PM
Hi,
The selction of a purifiaction and method depends on the molecualr weight and pI of a protein. Also, using ice cold ethanol, acetone, trichlroacetic acid, and fractionation by ammonium sulfate precipitation are all suitable for concnetrating proteins. However, using ice cold ethanol or acetone requires less work as compared to the ammonim salt method. However, if you want to retain your protein in an active form the use of Amicon or Geba mini-spin columns will be a evry good way to concentrate your sample. For the purification, if you have an antibody against this protein, then immunoprecipitaion will be the ideal way to purify your proteins, if this is the cas I will provide you with the procedure. Otherwise you need to start with FPLC first and then finish up with HPLC. Upon provide me with more information regrading your protein MW and pI I can send you an pproriate column for your fractionation and purification. Check this review as it may help you a lot EXS. 2000;88:29-42. Related Articles, Links Techniques for sample preparation including methods for concentrating peptide samples. Gevaert K, Houthaeve T, Vandekerckhove J. Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Universiteit Ghent, Belgium. Good Luck!
Re: protein precipitation after IMAC
Posted by: zia (IP Hidden, Unregistered user, )
Date: March 16, 2005 10:22AM
Respected,
i am great ful to you for nice suggestions. as much as concerned to immunoprecipitaion, i have a confusion. i m working the protein which have different amino acids sequences as sited by different literature. so the question is either one antibody will be enough for all proteins. second thing the amino acid sequence of such proteins present in plant is just 18-26. if one anibodies produes for all proteins its easy, but if seperate for each protein its too dfficult. second relating to hplc, as i told that the concentration is very minute (micrograms/100g) and different amino acid sequence, different mobile phases will be used and to work with such minute quantity, its more difficult. as no procedure is available for hplc. if you have any procedure related to hplc, i warmly welcome you. also send me protocole for immunoprecipitaion and anibodies production, as its my last step of work. regards Zia
Re: protein precipitation after IMAC
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: March 17, 2005 07:27AM
Hi Zia,
Please note the selection of any protein purifiaction approach relies on the physica and chemical proprteis of the protein. Is your protein is aresult of a proteolytic process? I have used the following IP procedure with great success 2.2.2.i Coupling anti-CrhC antibody to protein A immobilized on sepharose beads Coupling anti-CrhC to protein A was performed as described Harlow and Lane (1988). Briefly, 100 mg of lyophilized protein A immobilized on Sepharose CL-4B (Sigma) were swollen for 30 min at room temperature in 2 ml of immunoprecipitation coupling buffer (100 mM NaHCO3, 50mM NaCl, 1mM PMSF, pH 8.3). Anti-CrhC antibodies were mixed with swollen protein A beads at a final dilution of 1:50 and the mixture incubated at room temperature for 60 min with continuous end-over-end gentle rotation. The beads were washed twice with 10 volumes of borate buffer (0.2 M sodium borate, pH 9) followed by centrifugation at 1 000 g for 5 min. The beads were resuspended in 10 volumes of borate buffer, for crosslinking anti CrhC antibodies with protein A solid dimethyl pimelimidate dihydrochloride was added to a final concentration of 20 mM, and the mixture incubated for 30 min at room temperature. The reaction was stopped by washing the beads twice in 0.2 M ethanolamine pH 8, and then incubated for 2 hr at room temperature in 0.2 M ethanolamine with gentle mixing. After the incubation period, the 0.2 M ethanolamine was discarded after pelleting the beads by centrifugation for 1 minute at 14 000 rpm. The beads were resuspended in PBS with 1m M PMSF to be used for immunoprecipitation experiments. Good Luck
Re: protein precipitation after IMAC
Posted by: raksha (IP Hidden, Unregistered user, )
Date: April 8, 2005 04:29AM
hi
if u use urea in the binding buffer then the proteins will get precipitated.
Re: protein precipitation after IMAC
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 12, 2005 08:12AM
Hello,
Never use urea in your coupling buffer or SDS, you need to remove denaturing detrbents from your sample before applying it to the column otherewise the antibodies themselves will be denmatured and no IP is obtained. Good Luck!
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