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In this experiment, we transformed a truncation of the NFAT protein sequence into a plasmid vector to be expressed in E.Coli as a fusion protein with GST. We also attempted to transform the normal plasmid without NFAT so that we could get GST alone. After growing up these colonies and selecting positive colonies, we lysed the e.coli. We collected some of this lysate for SDS-PAGE. We then put the lysed GST and GST-NFAT solutions in separate columns with glutathione beads. GST binds glutathione. We washed the column and collected some of the wash for SDS-PAGE. Finally, we eluted the GST and GST-NFAT off the column and saved some for SDS-PAGE.

I have attached a picture of a successful version of the SDS-PAGE. enter image description here

You want to direct your attention to the first lane, the Molecular Weight Marker (MWM). The rest of the lanes should be interpreted in sets of 3. The first of the three is the crude lysate, the second of the three is the first wash, and the last is the eluted protein of interest. The important lanes for my analysis are 8-10 and 11-13.

I will explain what I know from this SDS-PAGE gel so far. I know the weights of the different bands in the molecular weight marker because I was given a copy of the different proteins in the marker. From this information, I was able to confirm that I isolated my protein of interest in lane 13 and isolated GST-alone in lane 10. I also understand the reason why the lysate lanes are darkest, followed by the wash lanes, and then very clean protein of interest lanes. Also, note that the marker and the elution lanes were loaded with 10 microliters while the other lanes were 2 microliters each. However, there are some things that I do not understand from the gel.
(1) Why are the bands so diffuse/low resolution. I imagine it may be overloading the wells, but I am not convinced because I followed a set procedure.
(2) Why is there large amounts of streaking, especially in lanes 5, 8, 9, 10, and 11.
(3) I isolated my protein of interest because it was a fusion protein with GST (or just GST alone), and then eluted the protein off the glutathione beads via excess glutathione wash. Theoretically, the lanes for the elutions should be free of everything except the protein of interest and glutathione then. This mostly holds true for lanes 4 and 9, which should only have GST and glutathione. However, lanes 7 and 13 should only have GST-NFAT and glutathione. The lanes are not as clear however and seem to have stained numerous other proteins. What is going on?
(4) I was told by a professor that the second band in lane 13 (the GST-NFAT lane) was a degredation product. How can this be? The band does not correspond to the weights of NFAT alone or GST alone. Also, if the NFAT-GST was degraded, wouldn't there be two bands of degredation product?
(5) Why do the GST-alone lanes have what looks like doublet-bands? (Lanes 4, 10).
(6) If we eluted the proteins of interest with excess glutathione, which has a very low molecular weight, why doesn't it show up on the gel? Is it possible that it ran off before everything else?
(7) I have a list of the weights of the bands of the molecular marker. Yet, there is an extra band at the bottom that does not show up on my list. Is it possible my list is incomplete, or is there something else happening?

Finally, I have attached a picture of a failed SDS-PAGE gel. enter image description here

The only thing that I definitively know was a source of error is the fact that my buffer level fell during the run (due to a leak). The results of this gel are accurate (Lanes 2-7 correspond to lanes 8-13 on the above gel, and the results are almost identical) yet the gel turned out horribly. How can the buffer falling cause this result? Or is there something else that may have contributed to this weird pattern? Finally, the lanes are not as distinct in this gel as the first gel, yet the same comb, and thus the same size and spaced wells were used. So why was there so much bleed between lanes/ lack of space between the lanes?

Thanks in advance for any help.

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  • $\begingroup$ These are a lot of questions and there is still some information missing to give you at least some answers. 1. Which molecular marker did you use (it would be at least important to know the bands), it would be even better if you could number lanes and buffer in the gel. 2. What did you put in which lane? 2-4 is crude lysate, 5-7 the first wash, 11-13 the elution, but what is in 8-10? $\endgroup$ – Chris Feb 26 '16 at 8:31
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Here are some thoughts based on my own experiences purifying many proteins and running many SDS-PAGE gels:

(1) Resolution--how fast did you run this gel? Often if you run at higher than say 150V or so you can get bands that look like this. The resolution could also be related to..

(2) I would bet the streaking is due to salt in the sample--a lot of the time lysis/wash/elution buffers can have upwards of 500 mM or 1 M salt, and this invariably causes streaking. Load less sample in these lanes and keep the total volume the same to dilute the salt below 250 mM at least. Other things in your buffers could be causing the streaking, but salt is the most common (let us know the composition of your lysis/wash/elution buffers).

(3) Your purification of GST alone looks very good--often your preps will look much more like the lanes you have indicated, which are not entirely clean. This is quite common for a one step purification--often you need 2-3 for certain proteins to completely remove contaminants. There are several possible sources of these bands:

  1. Endogenous proteins: Many bacterial proteins will non-specifically stick to the beads (this is why we wash), but even thorough washing can still leave some contaminants. These proteins also could be bound to your protein of interest and are co-eluted off of the resin.
  2. Truncation products: Depending where your tag is (let's sat it is on the N-terminus), you can sometimes get incomplete translation products--they have the tag, but they are not full length proteins. These will still bind and elute from the beads because they have retained the tag. Ignore this point if you tag is on the C-terminus, as in that case the tag will only be on full-length protein.
  3. Degradation products: This is related to your later questions--often during the purification your protein will be degraded by bacterial proteases. Did you include a variety of protease inhibitors in your lysis buffer? Keeping all of your samples on ice is also important for this reason.

(4) Understand that degradation products do not mean you get only your protein and the fused GST--they can be a variety of truncations due to degradation by proteases.

(5) I would bet this is just due to your running conditions or the salt in your samples. I think this is probably actually your single GST band.

(6) Glutathione will not show up on the gel--it's formula weight is about 300 g/mol if I recall meaning it's about 300 Daltons, so much smaller than anything that you'll retain on the gel. Further, remember than your stain is for proteins--Coomassie blue binds basic residues on proteins.

(7) I'm not sure which band you are referring to, but I think the very bottom band in your marker lane is simply the dye front. It could also be a degradation product of a protein in your ladder.

As far as the second gel goes--it is all due to the buffer level falling during the run. Without buffer covering the top of the gel there is no current flowing. Probably as the leak was occurring you had uneven distribution of the buffer. Strange things can happen when this occurs (unscientific, I know).

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  • $\begingroup$ Thank you for your detailed analysis. I have taken the information and used it to help me understand my gel more. (1) I ran the gels at 120V through the 4% stacking layer and then 200v through the 12% resolving gel. (2) I will look more into the salt composition and report back. (3) I still do not understand why the GST alone lanes were more free of extraneous proteins than the GST-NFAT lanes. (4) If a protease degrades a protein, would there not be two resulting products? Yet there is only one extra band. I attached a labelled gel to make things more clear. Thanks for the response! $\endgroup$ – PrinceAladdin28 Feb 26 '16 at 22:02
  • $\begingroup$ Definitely your streaking and resolution is due to the voltage you used--I usually do 80V for the stacking and 120V for the separating. You still get enough information running it that fast, but not great pictures (as you can see!). As for the extra bands--again these could be bacterial proteins bound to your protein (especially chaperones). I think this is the most likely issue--you can increase your washes to try and clean it up. The GST prep might be more pure because you have more GST than GST-NFAT--it could be fully saturating the beads, preventing bacterial proteins from binding. $\endgroup$ – user21630 Feb 26 '16 at 22:17

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