I'm working with two high molecular weight proteins:
- GFP-tagged Ankyrin-G (AnkG, 270 kDa + GFP = ~297 kDa)
- GFP-tagged NF186 (213 kDa total)
Both are expressed in mammalian cells via transfection. Under the fluorescent microscope, NF186 shows strong GFP signal, while AnkG shows fewer but still clearly fluorescing cells. So transfection, while not ideal for AnkG, is at least partially successful.
After lysis and Western blotting with anti-GFP antibody, I consistently get completely blank blots — no bands for either AnkG-GFP or NF186-GFP. I’ve tried:
- Semi-dry transfer: no signal
- Wet transfer at 100 V for 3 hours at 4°C: still no signal → (I used these settings because I have 50 kDa proteins in the same blot, which I didn’t want to lose due to over-transfer — so I was trying to balance large and small proteins)
- Ponceau staining shows no high molecular weight bands
- Blotting with anti-AnkG antibody also failed
- I also tried two different anti-GFP antibodies from different companies and species (mouse and rat) to rule out antibody issues — still no signal
Interestingly, in a co-IP experiment, when I pull down AnkG-GFP using anti-GFP beads and blot for a known low-MW binding partner, I get a strong band for the partner, confirming that AnkG was expressed and efficiently pulled down.
So the issue seems to be Western blot detection of the high-MW proteins themselves, not expression or pull-down efficiency.
Has anyone worked with AnkG, NF186, or other proteins ~200–300 kDa? What transfer conditions and antibodies have worked for you to detect such large proteins reliably?
Hello Farah,
I don't have an exact solution to your problem, but I've had similar issues with large proteins of interest.
Did you observe high-MW proteins in your lysate after Coomassie Brilliant Blue staining of the SDS-PAGE gel?
I had trouble isolating high MW proteins using the non-denaturing lysis method with non-ionic Triton X-100 detergent. My protein of interest is 175 kDa and carries two tags: GFP and HA (total >210 kDa). When I lysed under denaturing conditions (with 6 M urea and 1% SDS), I have successfully extracted high-MW proteins, checked by Coomassie staining, and detected my protein of interest using western blotting with both anti-GFP and anti-HA antibodies. I would suggest checking your lysate profile by Coomassie staining of the electrophoresed gel to see if you can extract high MW proteins. These high-MW proteins tend to aggregate and are difficult to extract using mild lysis conditions.
The wet transfer method is better for transferring high MW proteins.
We use an anti-GFP antibody from Santa Cruz Biotechnology Inc. (GFP (B-2): sc-9996), which works well for us.
A co-IP experiment could produce a positive result due to non-specific binding.
I am sharing one of my blot results and a Coomassie stained gel to show the lysate protein profile using a lysis buffer containing 6 M urea, 25 mM Tris-HCl at pH 6.8 and 1% SDS.
Best wishes,
Dear Farah,
Don’t Worry you can easily resolve these problem.
3 major step:
1- Use low percentage resolving gel like 5 or 6% (Use gradient gel 4 to 8%) for optimisation and increase the length of the resolving gel by maximum decreasing the stacking gel.
Reduce the tris-glycine concentration and ensure the complete desaturation of the protein boil in beta ME for atleas 10-15 min.
Run the gel till your Low MW protein (40mwt ) appears at the bottom.
2- Always use wet transfer 25-30 v overnight ( at least 12-16 hrs) and use only PVDF membrane (.2um), make sure membrane should be charged in methanol for 10 min.
For blocking, use the 6% BSA instead of milk.
3- Try primary antibody incubation for overnight at 4 degree and lowest speed on rocker.
Let me know if anything els.
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