Some problems are often encountered during protein purification. Here are the problems encountered in the following protein purification experiments and their solutions.
1) I now have a fusion protein on hand. When I was purified, I used GST affinity chromatography after dissolution. After cutting with protease, I found that the target protein was not active.
What are the possible reasons for this? How to solve it? Measured the gene sequence, no problem. After the cells are broken, the fusion protein is in the sediment. After I extract it with the cosolvent, I can use GST for affinity chromatography, but the efficiency is only about 50% to 60%. The elution protein does not require a co-solvent, but under such conditions, the target protein will precipitate after cutting. I can dissolve the fraction with 0.5% CHAPS and the target protein is more hydrophobic.
Since it is very hydrophobic, you can add some glycerin or ethylene glycol to reduce the polarity, so the dissolution will be much better. In addition, you can also directly add 2% PEG to reduce the polarity, while protecting your protein, you can do it again. Purification is fine. I used to use such a protein with 10% ethanol plus 3-5% PEG-5000. In this case, the protein still has high recovery rate. I think those surfactants can be used or not. Well, if you lose activity, you can monitor the extraction, purification, and enzyme digestion where it loses activity, which makes it easier to solve your problem. Also pay attention to the pH of the buffer and see if changing it has no effect on solubility. If the protein has a precipitate, you can slightly lower the concentration of the protein. This is also a solution.
2) Is there any affinity gel that has been synthesized with FMN?
Affinity fillers have been synthesized over 20 species. Do you want to use it to purify a certain dehydrogenase? I think FMN can be coupled with limited, but FAD has a good amino coupling, not to mention that they are almost the same coenzyme. Are you considering connecting the FAD? Of course, the structure of FMN also has a secondary amine that can be coupled to an epoxy-activated filler with a long arm, and the cyanogen bromide activation or other activated medium is not very good, so I think this is no problem.
In addition, if FMN is used as a coenzyme, I can also suggest that you try blue agarose gel because its ligand structure is similar to that of FMN, which can purify a lot of dehydrogenase and kinase. .
Corresponding coupled materials, to self-synthesize affinity fillers, there are many companies that provide a good medium for activation, and they can be coupled by themselves. Among them, the common cyanide agarose gel of Qiwei Yicheng Co., Ltd. Oxygen agarose gel, amino agarose gel, carboxyl agarose gel, activated ester agarose gel, and sulfhydryl agarose gel, etc., but if it is a small molecule, it is preferable to have 3-10 carbon as a ligand. The activation medium of the arm is good. In addition, there have been reports in the literature that when the coenzyme is immobilized, the coupling position is different and the selectivity is different, so you can choose your own suitable activation medium.
Different activated media have different requirements for the coupled ligand, so a clear understanding of your ligand can be used to select the appropriate activation medium.
I generally choose epoxy agarose gel in the synthesis, it can be applied in a wide range of amino-decyl hydroxyl groups, and the synthetic medium has good rigidity and less non-specific adsorption, so no special treatment of unreacted groups is required. In addition, the bonded bonds are stable and the ligands are less detached. I think it is a good choice.
The protein renaturation of inclusion bodies is not much, but I remember that a buddy said that the most useful method is also the easiest method. He said that when trying to refold, try not to think about too difficult technology, those are fashionable but not necessarily good. Used, commonly used dialysis renaturation, dilution and renaturation, including foreign experts also said. I feel that sometimes it is not necessary to start to touch it. The key is to change the conditions frequently.
Chromatographic renaturation is very fashionable, but there are not many that can be used. I think that the difficulty of protein is that most of them are different, so the key is to look at the literature and ponder over the characteristics of your own protein.
3) When the Sephadex G-25 (medium) column is desalted, does the salt concentration have too much effect on the efficiency of the column? If so, what is the general limit on the salt concentration?
You are welcome, demineralization should also have certain limits on the concentration. The same sample has a higher salt concentration than the lower concentration of the peak on the column, and the resolution of the column is higher. Point, but in the normal range, such as 1-2M should not affect much, but to be very clean or try to do less sample, I think the volume of the load is more than the concentration effect.
4) My protein is about 17kd and can be affinityd with heparin. It is usually purified by ion exchange and affinity chromatography. Now I am modifying it with polyethylene glycol. The molecular weight is increased by 5000, and the modification rate is unlikely to reach 100%. What purification method can be used to separate the modified and unmodified? (Of course, after the modification, polyethylene glycol remains, and this small molecule is also removed.)
Most of them use gel filtration. I think this is also a good practice. After all, PEG is a chain of molecules. It does not behave like a normal protein on a column. The higher the degree of modification, the later the peak, so your protein chooses sephadex. Try G50, its range is 1000-30000, it should be a good choice. In addition, PEG is a hydrophobic chain, and the modified protein has enhanced hydrophobicity. The higher the degree of modification, the stronger the hydrophobicity, which can be separated by this principle. Ion exchange can also be chosen, because the same reason, the higher the degree of modification, the more the charge is shielded and the less the charge, so the higher the degree of modification, the first. Affinity is also the same as ion exchange, you can try, the affinity of your hand can not be separated, you choose to see which is more economical and easy to use in these methods.
5) I have a problem for a long time, extracting enzymes from bacteria, as if using conventional methods (ultrasonic wall breaking, buffer extraction), I can't always get the protein I want. Is this protein also hydrophobic, need to add solvent to extract? In addition, can I also add some glycerin or PEG to extract?
In fact, I think this is the case. Since you are extracting, there must be precipitation and supernatant. You should know the molecular weight of your target protein. Then run the electrophoresis to see if it is in the supernatant or in the sediment. The next question you solve is how to extract.
For different enzymes, depending on how stable the enzyme is, you can use different pH to extract. I used to extract an enzyme that can't be extracted with water. It needs to be extracted with salt. Try it more and design a solution to solve the problem. So you don't have to add glycerin to use it. You have to pay attention to whether there is a problem with the broken itself. If you go back and do it, you may find the real reason. Any questions continue to be explored.
6) Is HPLC used for analytical detection or separation and purification?
If it can be used for purification, what is the significance of the small amount of sample loading?
If it is used for analytical testing, how to guide the subsequent separation and purification work?
HPLC can be used for both analysis and preparation. The amount of purification is small, so that the separation effect is good, and it is possible to obtain a very pure substance. At the same time, mass spectrometry can be used to obtain many single protein characteristics including sequences, etc. The fraction can not be obtained by general purification methods. HPLC has good reproducibility and accurate quantitation, so it can also be used for quantitative and qualitative analysis. It is an indispensable tool in analysis.
After the analysis, if the material is stable, direct linear amplification can be used for large-scale preparation. Therefore, the conditions for the analysis are met accordingly.
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