I did my gel running and transfer like i always do but this time when i stained the nitocellulose membrane with ponceau , the protein sample lanes apeared different towards the end . They appeared a little melted .
Could someone suggest why this could have happened.
Regards
Hello,
With Ponceau, you are staining the transfered protein on the membrane. Did you noticed a similar pattern of protein bands and marker while running? Did you stained the gel with coomassie after transfer and then checked in it ?
It would be better if you put some pictures .
Regards
Pranav
Hey,
i did not do a Comassie, i usually never need it.
This has never happened before .
I am attaching a picture below and you can notice the strange melting pattern in the lower half of the blot.
Cloud be diferent things:
1. Wrong running buffer (or gel buffer). Resistance of the gel increases. Gel heats up.
2. To high Voltage/Amperage
3. Gel not completly polymerized
Point 1-3 are possible but unlikely since your marker looks fine.
4. Problem with sample buffer
5. to high salt concentrations in the samples
6. Problems with lysis
Point 4-6 more likely. Only your samples are affected.
Best
Karsten
Hi Pranav,
Could be a lot of things, but my first guess is that there's a high salt/SDS concentration in samples at the right of the gel. Other thing it could be happening to those samples is that they have high quantity of lipids.
Good luck,
Valentín
Hello,
I agree with the above comments. Your molecular weight marker looks fine so, I would suggest you to start with changing the loading buffer/dye.
Best luck.
Pranav
Thanks everyone for the help. I got rid of the problem today .
I think it was a mixture of old acrylamide , TEMED and Tris buffers.
Thanks a lot
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