Hello

first of all can I know the name of the person ask this question?

Secondly you have sent the electrogram without write the samples of each slot refer to what??????

I think you need to read the SDS electrophoresis : its principle, uses and how to explain the results.

There are many books and booklet deal with this subject i.e. many volumes of the series of Methods of enzymology Vol.1 Vol.184 moreover there are many booklet of LKB company as well as Pharmacia company you even can find them in the internet .

I agree with Hathama Razooki Hasan

It looks like you are trying to purify a particular protein about a third of the way from the bottom. Lane 4 looks like a good amount of purity that still retains a high concentration, but it is difficult to conclude anything else without more information.

You can use the molecular weight markers to measure the approximate monomer molecular weight of the purified protein.

You can use densitometry to measure the purity of the protein (although the staining is a bit light in the photo).

Let's see. Inotice very clear zones on your gel. Does this mean you've had troubles with the gel?. It is 5 lanes plus the markers. Withthose markers you may determine the size/weight of your protein, as heavier proteins do not travel as long along the front. (It depends but you may get any). Those markers must have a molecular weight. The bands may be of different size, but this can be mended, ImageJ, densitometry... Alinear regression can be established with all the information. Maybe suggest it could be a buffer. Is there a particular reason for one of your proteins to be showing? It depends on what you want to do with it. Did you loose precision (SDS)? The protein has gone through a process... The purity can be different, but maybe (maybe) it would have been pure otherwise. Homogenity? What has been the previous steps? Do you need to recover protein? What are the proteins? Could there be more than one? Two? It will depend on what you call purity. Always the same band on all four samples? (The same mass with more "purity"?) The bands are thinner (purification)

Your Name

I guess that if your GFP is the more intense band that is recovered in the eluted fractions, you could try to increase the amount of resin used for the purification (NiNTA?). This is because there is a lot of protein going away during the washing step and there might be a decrease in 30-40% (I'm just guessing without knowing the volumes, protein concentrations and so on) in your yield. It might be also a problem of composition of the washing buffer, though.

You should have loaded the flow through of the purification, since it would have helped to see if the problem was the amount of resin (so the total capacity of the system) or the composition (ionic strenght, pH, detergents and so on) of the wash buffer.

Your Name

It looks like you purified a lot of GFP and you also lost a lot of GFP. Could be because your wash buffer composition is 20 mM Imidiizole, etc ... If the His-tag binds weakly to the Ni-NTA beads then you are eluting your protein in the wash buffer. Wash it with 5 mM Imidizole or even 0 mM Imidizole might be required.

Next time you do the Purification. reclean the nickel beads before use.

6 M Urea, 100 mL to dirty Ni-NTA beads to unfold all proteins stuck to the beads. Wash the column with copious amounts of water to get rid of the Urea, Strip nickel from the beads with 100 mM EDTA, 200 mL. Wash out residual EDTA with 400 mL of water. Recharge the NTA beads with Nickel using 100 mL of 100 mM Nickel Chloride or Nickel Sulfate. Store recharged Ni-NTA beads in Ethanol to prevent microbial contamination.

Use more Ni-NTA beads next time and less Imidizole which I presume you actually have in your wash buffer.

You have purified the protein already.(3-4th well).U need a stand mol wt marker to compare. If u do not know about this protein.Just cut the band send it for MALDI-TOF.

If you get multiple (more than one) band in SDS-PAGE, you might not then be able to judge your sample purity and may need to do native-PAGE for that purpose.

I think that you need to concentrate your sample before runing the electrophoresis with cold acetone, try to remove the not specific protein ( ultrafiltration n'y centricon tube) and chose witch fraction contained your protein.

The best procedure to confirme or to determine that with mass spectrometry

I think these 2 articles (attached) could actually help you.

Best wishes