I eluted purified his-tag protein in 200 ul of elution buffer containing 500 mM imidazole. Then, I want to use it for SDS-PAGE to confirm the presence of specific his-tag protein band on gel. What is the proper step before using this eluted purified protein for SDS-PAGE? Is it ok if I directly use eluted protein to be mixed properly with sample loading buffer for running eluted sample in SDS-page? Any helpful comments? Thank you.
To load protein in imidazole, it is preferable to add loading dye and not heat it at higher temperature. It can be kept on water bath set @37 degree Celsius. This protocol works fine and the proteins also gives crisp bands.
Ok,thanks..But,.is there any possibility of having low-molecular weight of protein contaminants after running SDS-page if my purified his-tag protein still containing imidazole? My predicted MW of desired protein is 56.1 kDa.
For simple gel electrophoresis you do not need to dilute out Imidazole; however, if you going to do any functional assays with the recombinant protein you will need to dialyze the protein solution.
You can mix the elute with the sample loading buffer, and heat denature at 95 for 5 min, and load it in gel. No need to remove imidazole.
I usually load know amount of protein to check for purity. Usually 2-4ug of purified protein and standard in case you have one.
For protein sample containing imidazole, which method is better, either heating it at high temperature (i.e 90-95 degree celcius) or low temperature (i.e 37 c)? The protein will be mixed with sds sample loading buffer subjected to sds-page. How long can I heat it, either 3 minutes or 5 minutes?
I do it for 5-10 minutes at 95. I would stick to 5min at 95.
The protein is pretty much stable in Laimelli buffer (Sample loading buffer).
Sorry forgot to mention. There is the inversely correlation between denaturation time and temperature. At 95c it's quick and 5min is sufficient to completely denature the protein.
Cheers
If I want to keep that protein after sds-page, what is the proper condition for protein storage? I mean, storage buffer and temperature. One more question is, how to run functional assay if I use purified protein that still containing imidazole? Do I need to remove it using Hi trap column or dialysis tube?
For protein storage, the condition of choice depends on the stability of your protein and on the duration of storage. In general, try not to use storage buffers of extreme pH, and of pH values that are close to the pI of your protein. Most proteins should be fine to be kept at 4C for short term storage (within 24 h). For long term storage, it is necessary to keep the protein at -20C, and you may consider adding 50% glycerol to avoid freezing. In most cases, the presence of imidazole does not interfere with functional assay, but in my experience, leaving protein in imidazole can cause protein to precipitate easily. So, I always remove imidazole from the protein prior to any downstream manipulations. Imidazole can be removed by dialysis, ammonium sulfate precipitation or ultrafiltration. I think that a centrifugal filter unit is especially useful for both buffer exchange (into storage buffer) and imidazole removal.
Yes you can do SDS PAGE with eluted proteins right after the purification, just add the classical loading buffer (with SDS) and denaturate your sample (5min/100°C) . I produced/purified two His-tag insect enzymes and I never removed the imidazole. Then I did enzymatic tests and it worked just fine. It really depends on what you want to do with your purified proteins.
For storage its depends on the stability of your protein. One of my proteins was very stable (I kept it at -20°C for months with 50% glycerol and it was still very active) when the other one was much less stable (I kept it at 4°C for maximum 24h but most times I did functional tests right after the purification).
It is better to heat the samples to no more than 37 C for 5 mins in the SDS loading buffer prior to analysis. Heating a sample that contains imidazole at 95 C can lead to acid labile bonds to hydrolyze.
You may refer to this article:
Crowe J, Döbeli H, Gentz R, Hochuli E, Stüber D, Henco K. Methods Mol Biol 1994;31:371.
[PubMed: 7921034]
I got good information from the site for storage, remove the imidazole from the purified protein. i accept the suggestions of Dr. Nicolas Durand.
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