Hello
I have been trying to express and purify two truncated his-tag proteins(45KDa and 65KDa size without his-tag) using baculovirus expression system. I was however able to express the proteins using SF9 cells but when it comes to purification, it was not successful.
Initially I used Nickel chelating resin to purify the lysate and used the eluate to bind with another protein (46KDa). This I tried purifying using Hydroxylappatatite column since my 46KDa can be purified using this column. I then, tried Sephadex G-75 on a longer column to do size exclusion to get the complex. Before adding it to the sephadex column, I can see the protein in SDS-PAGE and could confirm it using western blot. But after size exclusion, I cannot find my protein in any of the samples but my complex forming protein (46KDa) can be seen.
I thought since I have been losing protein at every stage of purification, I wanted to reduce steps. I now directly added 46Kda to my protein after the Ni- purification step and then loaded onto the size exclusion column, but the result is still the same.
Can anyone tell me what could have been happening?
Did you concentrate the protein before to inject to FPLC? It is very important.
How many ml did you inject? In size exclusion chromatography it is also very important the volumen that you inject. This volume must be indicated in column instructions
Other option, you could increase the time of imidazole gradient (less pronounced) in the affinity chromatography, in order to obtain a better separation. Perhaps, after this you would not need another extra chromatography step and you could remove the imidazole using Amicon Ultra-15 Centrifugal Filter Units
Thank you for response.
I concentrated the protein before adding onto the column.
The volume I used was 2ml and the column volume is 500ml. I tried doing imidazole gradient and I lost little of my protein every step.
But can you see your protein on the computer screen at 280 during the exclusion size chromatography? Have you calibrated your colum in order to know the minimum and maximum elution volume?
Perhaps you could replace the exclusion size chromatography by an ionic exchange chromatography. You can calculate the Isoelectric point of your protein using http://web.expasy.org/compute_pi/ and after this you can use a cationic or anionic exchanger depending of the pH (eg High Q of High S cartridge from Biorad).
Thank you so much. Yes, I can see a peak during size exclusion chromatography. I thought may be the protein was so diluted that I am not able to detect it in SDS-PAGE staining, so I performed western blot and could find traces of my protein in the samples but very little.
I checked the PI of protein and I will try ion exchange chromatography.
Thank You
Perhaps this is the problem, so you can take all the aliquotes of corresponding peak, mix them, and concentrate 5-10 times.
In addition chek that your protein is not precipitated, because His-tag could make your protein insoluble in aqueous buffer.
Thank you so much. I am already concentrating it. How do I check if my protein is getting precipitated?
Dialyze your protein 5-10 times to remove the imidazole (replacing imizadole by your storage buffer). After this concentrate the protein (e.g. 2-3 mg/ml) and leave it at 4 ºC a few hours. Finally, centrifuge 1 min at 9000-10000 and check it if the protein is precipipitated (look at the bottom of the eppendorf)
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