I am trying to purify my protein from the cell culture supernatant. I transfected my cells with my construct (which has a double his tag) to produce my protein in the supernatant. I collected the supernatant about 1 litre and after dialysis I subjected my supernatant to purification but I don't get my proteins at the end. Do you have any suggestions?
Dear Gautam,
Yet it is not very common, but sometimes during folding of proteins, his-tag might be folded inside of protein and then it cant recognise Ni-resin. You can change the tag to another end, from C to N terminal, or vice versa. It definitely works. But why are you doing dialysis of protein expressed media? If it is just normal medium, may be you can reduce the serum 2-5% during expression and then just dilute the whole media 2X by addition of buffer (50 mM Tris-HCl, 20 mM imidazole, 500 mM NaCl, adjust final pH 7.5) use concentrated stocks like (3 M Tris, 2 M imidazole and 5M NaCl). After maintaining pH, just add 2-5 ml fresh Ni-Resin and role over the bottle at 4 degrees overnight. Next day keep the bottle standing for 3 h so the resin will settle down. now you can decant 900 ml media from top and pack resin in column (use can use mild peristaltic pump to suction for accelerate packing). Now wash the resin and elute the bound protein. This method I used for more than 50 proteins expressing in cell lines. If you are using DMEM for culture, then select DMEM advanced.
Second if you are sure that your protein is expressing in good quantities, you can precipitate them by Ammonium sulphate and redissolve them and purify by ion exchange. Then you should use serum free media during expression, becuase serum will interefere in your ammonium sulphate pptation.
1. Find pH stability window of your desired protein. what is the size & pI of your protein.
2. Do dialysis or Diafiltration of supernatant ( Take 3 times lesser MWCO of membrane ) & filter it to reduce turbidity from your output.
3. You can use metal cheating affinity chromatography as per Mr.Birendra singh , But if that is not work out than go for ion exchnge chromatography as per pI & pH stability window of your protein. Screen the salt gradient , Fractionate it & Analyse it. Its definitely works.
Best luck
The first step is to identify that your cDNA insert is in frame. A simple test using in vitro coupled transcription&translation in the presence of 35S-methionine will give you the confidence that you have the accurate starting clone. Analyze the translation product by SDS PAGE and autoradiography.
Secondly, establish a stable cell line expressing your protein using selectable markers such as G418 or puromycin. Choose 4-6 colonies resistant to the drug and grow them in 50ml flasks. Choose the clone expressing high levels of your protein. Start a pilot experiment with 10ml medium containing various percentage of supplemented FBS. Remember that there are serum free medium in the market which you can adapt for your specific cell.
Since by this time you know that the starting material is well established, you can start collecting medium from large cultures. Instead of dialysis and freeze drying try ammonium sulphate precipitation to concentrate the proteins in the medium. You may need 50-60% ammonium sulphate.
After concentration, you can dialyze to remove the salt and perform affinity chromatography on Talon affinity matrix.
Remember to siliconize all glassware and plastics used in the purification. Proteins tend to stick to walls of the tubes and glassware. You should follow the purification by analyzing samples by SDS-PAGE and Coomassie Blue or Silver staining.
The short cuts works sometimes but it is risky with projects of importance.
I have 40 years of protein purification experience. I still continue my research and I enjoy bench work. There are certain part of the laboratory procedures which can be transmitted, however some intricate techniques you learn during the development of the process. For example, a receptor protein which is in micro g/g of tissue to be purified in its native state and with out degradation you need to view the stability, degradation and analysis of function in every step of the work.
My suggestion to you is to be systematic and methodological as possible. You will definitely make mistakes and learn from them. You have to be critical of yourself and focus on your aim.
1) Have a thorough understanding of the molecule you want to purify such as its function, molecular mass and stability.
2) Analyze the protein in its native state by conventional chromatography such an ion-exchange, gel filtration etc.
3) Develop techniques to study the nature of native proteins functions. This will provide you with clues which can be used in purification.
4) Always start pilot experiments before big endeavour.
5) Have routine techniques at hand such as SDS-PAGE, immunoprecipitation and Western blotting.
6) In eukaryotic and bacterial expression start with a known candidate to gather information and experience.
7) Develop strict protocols and follow them every time.
8) If your protein can be purified by affinity techniques, from a diluted solution you need to develop methods to enrich it before purification. By analyzing the flow-through of the columns, you will know whether the matrix retards your protein.
9) Unspecific absorption to the affinity matices can be eliminated by adjusting the salt concentrations and by including different detergents in buffers.
10) For example if your protein is an enzyme, you may be able to test its activity in bacterial suspension before purification. The levels of expression can be analyzed in crude extracts by Western blotting.
Wish you best of luck
Dr. Manjapra Variath Govindan
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