I am using a bacterial protein and its get aggregated. I get a clear band of appropriate size in SDS PAGE but unfortunately it forms smear pattern in NATIVE PAGE. Also i try to check in TEM and found several aggregates.
Proteins are purified using Flag Affinity Purification, followed by gel purification.
pI of my protein is 7.28 and buffer I use is TBS of pH 7.4 (at 4C)
The chromatogram from your gel filtration step is a source of information regarding protein stability and a well folded protein, many scientists focus too much of SDS-PAGE gel, in fact often just using SDS-PAGE for selecting fractions instead of using both.
GF buffer selection is a part of good gel filtration practise as the protein will be in that buffer for a considerable amount of time in this environment.
I don't like Tris as it is not really a good buffer. For GF I generally use 10-25mM HEPES 7.5 (in your case I would use pH 8.0), with 125-500mM NaCl, 5% glycerol and 0.5mM TCEP (non-thiol reducing agent with v. good stability).
Hi, what does the chromatogram of your gel filtration look like, a nice symmetrical peak or something else?
Also, what is the gel filtration buffer?
I would have a look at papers where similar or ortholog proteins have been purified and use the buffers that have been established as suitable for trialling stability/solubility as a starting point.
If you have access to a suitable RT-PCR machine you could try a Tm shift assay to establish which buffer is best from a screen
Would help if the SDS-PAGE gel and Gel filtration images are shown.
A buffer screening suggested by Stanley Ng is a good idea..
Is your fusion protein eluting at the appropriate elution volume or is it eluting at the void or higher than expected on gel filtration..?
Hope there is no free cysteine - if so add DTT (2-10 mM) or TCEP.. Or add more NaCl (300-500 mM), glycerol (2-5 % or more) and so forth..You can try a number of buffer conditions and run on an analytical gel filtration column to confirm if it is eluting at the estimated elution volume or so...
You may use tween 20 and CHAPS as a non detergent protein stabilizer or u may dissolve ur protein in a stabilizing solution of urea and CHAPS.
In case of tem PEG(polyethylene gycol) acts as good stabilizing agent for better imaging..
Is your protein very hydrophobic? Which gel purification do you use? If you are using an affinity chromatography step, remember that this kind of chromatography concentrates very much your protein after elution and protein concentration is an important factor in aggregation, the pH of your buffer also is very close to the pI of your protein, you should take into account all these factors, probably modulating buffer properties you could decrease in some degree the aggregation
This suggestion may not be relevant because you provided so little information about your particular protein, but as a general rule, a protein is least soluble at its isoelectric point (pI).
Glad for all your suggestions.
Please find here the gel filtration images on SDS PAGE and Native PAGE. I think solubility of the protein is the problem.
Stanley Ng gel filtraton isn't a problem and my protein showing correct MW . I will try to add Glycerol to my buffer as suggested by Bubacarr G Kaira .
Looking forward.
Hi with the limited information provided about the protocol used in native gel preparation I suggest to adding DTT or beta mercaptoethanol to your gel and buffer and see if this causes dissociation also check the pH of the gel and should be far enough away from the PI of your protein you can change the gel buffer and see if the bands sharpen because smears often caused by incomplete solubility furthermore if your protein is denatured by urea and loaded to the gel it might incorrectly refold and aggregated
good luck
The chromatogram from your gel filtration step is a source of information regarding protein stability and a well folded protein, many scientists focus too much of SDS-PAGE gel, in fact often just using SDS-PAGE for selecting fractions instead of using both.
GF buffer selection is a part of good gel filtration practise as the protein will be in that buffer for a considerable amount of time in this environment.
I don't like Tris as it is not really a good buffer. For GF I generally use 10-25mM HEPES 7.5 (in your case I would use pH 8.0), with 125-500mM NaCl, 5% glycerol and 0.5mM TCEP (non-thiol reducing agent with v. good stability).
Lot of good ideas above.
About solubility, stay clear from the pI. At least 1 pH unit away from pI would help improving solubility. As you are running native PAGE, I would assume you are interested learning about the native/folded state of the protein. For that, use non-denaturing (but solubilizing) buffer systems with low salt - such as histidine (or citrate) buffer at pH 6 with 250mM sucrose (or trehalose) and 0.1% polysorbate 80.
Hope it helps.
Abdelrahim Ahmad Hunaiti , Stanley Ng , Tapan K Das Thank you so much for wonderful suggestions. I want to look into native state of my protein thats why I did NATIVE after gel filtration. I consider based on the discussion, the primary problem might be with the Gel Filtration buffer i use. I will try next time with Stanley Ng suggested buffer by avoiding Tris. I also did SEC-MALS to understand folded state of my protein and it was too much worse result due to aggregation.
I will come back to conclusion as I purify my protein again keeping in mind the pH of my buffer and avoiding Tris. Thank you all again.
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