First was the gel prepared by you or industrial made? 12%?

I dont think the band indicated might be affecting the running. The bottom arrows I see should be dye front.

I also dont think the 25KDa indicated is your expected 17KDa protein even though it takes 3 h to run on 110V - I suspect the protein wasnt overexpressed or maybe it was expressed but present in the inclusion bodies (pellet of lysates). Was the lysates sonicated and/or lysozyme prepared before adding the SDS loading gel?

MANY possibilities;

Strain used to induce/overexpress.

Membrane protein are insoluble.

Why do you doubt the expression of my protein?

This paper suggests that Membrane protein MW shifting happens...

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2644111/

That band is just too strong compared to the other proteins to not be overexpressed , i will repeat a run tomorrow with an uninduced control and check , I'm also Ni-NTA purifying the sample in this gel

Dear Friend,

The band like appearance at the end is actually the wave front of the dye. When I was doing SDS PAGE (12%) for the smallest band of size 17 KDa which was my desirable band size I also took nearly 3 hours (2 hrs 45 mins) for a complete run. First of all I would like to suggest please do not run your gel continuously at such a higher voltage from start. Run initially at a low voltage of 60 to 80 V till all the samples cross the stacking gel and enter completely into the resolving gel. Also note that running the proteins at a higher voltage may heat up the gel and diffuse the samples reducing visibility of the bands. Talking about the 17 KDa band you should get; perhaps the denaturation step is not well performed and thus the monomeric bands are not found. Is the protein dimeric in nature in the extract? Because if the protein is a homodimer by nature you might be getting a 34 KDa band (not 25 KDa band as mentioned) due to improper denaturation (Dimers not separated into monomers).

Firstly my protein size is 21.6 not 17 KDa (miscalculation) , I did a Ni-NTA purification in the following manner:

Buffers: R: 100 mM NaH2PO4, 10 mM Tris–HCl, 8 M urea pH 8.0.

W1: Same but pH 6.3

E: Same pH 4.5

1) centrifuged 150ul of the Ni-NTA agarose supplied from Qiagen at 6,000  ×  g for 1 min. Remove the supernatant and add 150ul of R buffer. Mix gently then centrifuge it again. Discard the supernatant, wash again once more, and finally 75ul of buffer R.

2) Add 50ul of the slurry to 300ul of cleared cell lysate(lysed in buffer R by pipetting and centrifuged at 15000g at 10 min and supernatant collected) And mixed at 400rpm at 25C for 20min in a thermomixer

3) centrifugation (6,000  ×  g, 1 min). Discard the supernatant (took a sample for SDS-page).

4) Wash the Ni-NTA agarose pellet twice with 300 μL of W1 buffer followed by centrifugation at 6,000  ×  g for 1 min, discard the supernatant.

5)Add 30 μL of elution buffer (buffer E), and gently mix by pipetting. Centrifuge at 6,000  ×  g for 1 min and carefully transfer the supernatant containing the fusion protein into a fresh tube. Repeat the elution step once more.

So my protein definitely has 6x-His tag confirmed by sequencing

After suspecting those bands i did the above and got this Gel with the bands staying in the first supernatant and not binding to the Ni beads. Does this confirm those bands are not my protein or is my protocol flawed? Check Gel

The last 2 lanes are the supernatant from step 3 and they show the same bands just above the 25kDa marker

The next 4 lanes are 2 replicated of the first elution and the second elution with no bands

The seventh lane in which you can see some bands is the uninduced control which confuses me as to why the bands aren't as strong as the induced sample, does expression induction increase the expression of non-target proteins? My strong bad isn't present in the uninduced control but then again the other bands aren't as strong either

Assuming you run the usual 8 x 10 cm2 gels, you can start with a constant current of 10 mA and increase to 20 mA when the dye enters the separating gel. Constant current is quicker than constant voltage.

100 mM Phosphate is is quite a lot of salt, which may interfere with electrophoresis and may explain the wavy bands in your gel. Electrophoresis may work better after methanol/chloroform precipitation of the protein (Wessel & Flügge 1984).

The background of the gel is rather high, you should destain for longer. A bag with activated charcoal speeds the process, also some foams used for packing have a high affinity for CBB, a small piece is all you need each time. Also, warming (not boiling!) gel and destaining solution in a microwave helps.

Thank you Engelbert but it hasn't stained well because i stopped the process as soon as i saw the bands, it destains very well if i leave it for longer.

I ran an empty vector an uninduced vector with my protein and obtained the same strong bands so i guess i didn't get my protein... back to the drawing board.