Firstly see that your protein does not crash out of solution. After refolding, if turbidity is absent/ minimal there is a fair chance that soluble aggregates formation is less. The best way of proving that your protein has refolded correctly is to carry out a functional assay. Apart from that there can be some other ways in which you could establish refolding. Concentrate your refolded protein and run it through a size exclusion chromatography (SEC) column. If it comes out in void volume it indicates formation of soluble aggregates. Proper analysis of SEC data is an indication which can be backed up with other data later. For example if your protein is known to bind with some other protein then you could carry out SPR (Surface Plasmon Resonance) studies to establish whether your refolded protein is having binding capacity with its binding partner or not.

Another approach that can be done is to simply carry out a 1D NMR study. Looking at the proton chemical shift is a good indicator of whether your protein has refolded or not. For example in case your protein has not refolded, there will not be any peaks below 0.7 ppm chemical shift unlike in case of refolded proteins. There are a few other things to look out for in the proton NMR scan which indicate protein is refolded or not. In case 2D NMR data for properly refolded protein (which was also functional) is already assigned and available then you can match your HSQC data with that to conclude whether this batch of your protein has been properly refolded or not.

There are several ways to see whether your protein has refolded or not. But the most reliable way in which one can say that refolding has been proper is to check by some appropriate functional assay.

Hello,

a simple method would be UV-CD spectroscopy. If you have the protein available (in a folded variant), you can compare it to your refolded protein. This gives you information about the secondary structures (alpha-helix resp. beta-sheets).

Hi!

The best way to ensure the correct fold is running an activity assay. 

Firstly see that your protein does not crash out of solution. After refolding, if turbidity is absent/ minimal there is a fair chance that soluble aggregates formation is less. The best way of proving that your protein has refolded correctly is to carry out a functional assay. Apart from that there can be some other ways in which you could establish refolding. Concentrate your refolded protein and run it through a size exclusion chromatography (SEC) column. If it comes out in void volume it indicates formation of soluble aggregates. Proper analysis of SEC data is an indication which can be backed up with other data later. For example if your protein is known to bind with some other protein then you could carry out SPR (Surface Plasmon Resonance) studies to establish whether your refolded protein is having binding capacity with its binding partner or not.

Another approach that can be done is to simply carry out a 1D NMR study. Looking at the proton chemical shift is a good indicator of whether your protein has refolded or not. For example in case your protein has not refolded, there will not be any peaks below 0.7 ppm chemical shift unlike in case of refolded proteins. There are a few other things to look out for in the proton NMR scan which indicate protein is refolded or not. In case 2D NMR data for properly refolded protein (which was also functional) is already assigned and available then you can match your HSQC data with that to conclude whether this batch of your protein has been properly refolded or not.

There are several ways to see whether your protein has refolded or not. But the most reliable way in which one can say that refolding has been proper is to check by some appropriate functional assay.

A good method to check the correct folding of your protein in an appropriate buffer  would be UV-CD spectroscopy that gives you information about secondary structure (far-UV) and tertiary structure (near-UV). Basic prerequisite is an exact concentration determination. Compare e.g. the secondary structure of amino acid sequence by in silico methods with CD-deconvolution of your experimental spectra.

Nevertheless it is necessary to check the correct refolding with additional appropriate functional assays.

@Aditya Jyoti Basak and Stefan Gajewski

 substrate for enzyme assay which i have very limited, i can not do that to check the refolding of protein. :((

Hi Huong Nguyen Lan,

Your protein molecular weight 56 Kda and Insoluble expression ie Intracellular.

for insoluble protein you have to follow below route:

1. Lysis of cell pellet (check how much pressure is required to rupture the cells)

2. Crude protein washes, to remove membrane protein, host cell protein and related protein. 

3. Inclusion bodies solubilization with Urea/GuHcl and reducing agents BME/DTT, you have to find out pH and buffer capacity.

4. If protein is less cystein bonds is easy to refold but some time protein is having very high cystein molecule like Enterokinese difficult to refold becouse they have 9 cysteine. 

for better refolding protein must be 60% pure.

or after solubilization you can use IEX/IMAC column (depend on behavior of your protein) to purify the protein. 

for refolding analysis you can use as analysis: CD spectra,  for alpha helix and beta sheet how it is ?

HPLC is also simplest method to check refolding % and purity.

If you give more physical parameter about your protein, i will tell better way...

Hi Surya Bhushan Kumar

My protein has 11 cysteine, and i use 8M Guanidine and Reducing agent is DTT. I am using Thermo scientific Pierce Protein Refolding Kit. 

And i have another question that is : after solubilize inclusion body of my protein, i run SDS-PAGE for analysis , and actually, i can not see any bands. On the other hand, I  want to compare before and after adding DTT, but the bands of my protein were same size (56kDa) . Is this alright or the band should not be the same? 

11 Cysteine means one is open cysteine if cysteine 5 disulfide bridge and one open cysteine enhance aggregation very fast, i observed in many cases above also iam trying to say same thing like Enterokinase.

Using protein with 6-8M GuHCl and use Cysteine ie reduced cysteine.like 20-50mM.

for my understanding now you have 11 cysteine it is so difficult to refold if will give you 1-5% refolded protein if you go with correct refolding condition.

you are not able to see protein of interest band in denature condition,  it means protein is not solubilized fully add more amount of DTT or equi-molar concentration of  DTT to solubilized the protein fully.

first you have to try full solubilization, if it not soluble, you will not get refolded protein any time.

for solubilization used GuHCl, Cysteine, Tween (80,20) and buffer capacity concentration you can find out with protein concentration.

don't play near to its PI for solubilization.

after completion of solubilization you can try IMAC column in denature condition, here purity may archive 90% than go for refolding (with very low concentration) 

from your Question: Can not find bands difference in SDS_PAGE,  with : without / Renature; Denature bands (you can find difference in MS/MS, mass difference)

Do it it will work, but refolded protein/Native protein you can not expect very high because of 11 cysteine.

Compare the activity of the refolded protein with that of the same protein from related organisms. Result shall be quite similar. Also, CD curve of refolded protein can be correlated with the native protein if available. Lastly I would prefer one simple experiment before all these, do a size exclusion chromatography and find out if your protein is eluting at the exact position as expected, if not, then protein might not be folded completely..

Thanks for all of the responses. I will be back if i can get any result. 

 It sounds strange that after inclusion bodies solubilization you cannot see any band in denaturing gels. This could mean that your recombinant protein is not being correctly solubilized. Maybe you should try a different solubilization buffer.  

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