I used the pet16b containing an histagg to purify my protein of interest. Ni-NTA beads were used to purify the protein. after washing, the protein was still present when runned on the sds-gel .However when doing the elution the protein doesn't elute? what can be the problem?
Hi Abire,
There are several reasons:
1. There is a problem with the formulation of the elution buffer used. Please prepare fresh eluent before elution and pay attention to the concentration of each component.
2. I usually use 150mM NaCl to elute. Maybe your target protein needs to be eluted with a higher concentration of NaCl. Try 300mM NaCl but pay attention to protein aggregation.
3. If it is proved that there is no problem with the elution buffer but is still a large amount of protein on the resin after elution, it is likely that the target protein is unstable and prone to aggregation. It is suggested to construct another clone or replace the tag that helps the protein to dissolve, such as MBP. In addition, before the next use of the nickel column, you need to regenerate the resin.
Hope it helps!
1) you need to make sure that 100 mM EDTA is enough to completely strip your protein from your preferential metal.
2) you need to get data that the binding of your protein with preferential metal does not result in the protein precipitation under conditions used.
3) you need to check that your EDTA stock solution is fresh-prepared and has pH ~ 8.0.
4) depending on the method you use for your protein determination, you need to check if the binding of your protein to this metal or exposure (presence of) to EDTA does not affect the results of your protein determination.
5) you can completely elute your protein if, instead of EDTA you use some acid buffer with pH ~ 4.0, collecting the eluted fraction into a tube with some neutralizing buffer to get pH close to the physiological optimum for your protein function.
Mohammed Nader Shalaby my elution buffer doesn't include EDTA. Are you recommending me to include that? I usually use 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 250 mM imidazole, and 0.05% Tween 20 .
I am actually really confused why the buffer should include EDTA? EDTA as a chelator strips the Nickel from the Ni-NTA beads, thus making them useless.
So no protein purification from this way.
Maybe our Ni-NTA buffer compisitions can help you:
https://cube-biotech.com/purification-using-his-affinity-agarose
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running