Why am I getting this type of pattern?
I am thinking the problem is either the running buffer or the sample buffer. I have changed new running buffer but the bands are still crooked. Could the problem be the chemicals (SDS and Tris) since the dated year on the bottles is in the 1980s. What other problems can there be?
Thanks
Use new sample buffer. On the other hand, protein samples should be well disolved. Centrifuge these samples before mixing with sample buffer. Good luck
I forgot to say that electric current should be uniform through the gel. Try new glass plates for gel polymerization
Also make sure to add 1x sample buffer to unused wells (if you have not already done so)
Have a look at this troubleshoot http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gelgoofs/misc.html
Dear Christy
There are some possible reasons:
1. No appropriate protein solubilization
2. Dirty plate of gel casting
3. Existence of baubles in you gel
4. You gel was not polymerized appropriately.
5. The electric current may be change or your gel may be too warm.
6. The salt concentration is too high
7. Excessive pressure has been applied to the gel plates when the gel is placed
Since these are pre-made gels, polymerization and gel quality should not be issue (if they were stored properly, and not too old). To me it looks that your casting system is not so good, so when you put the gels into the casting (squeeze thingy) not equal pressure is applied on the gels. Additionally, some of the wells (middle ones) might get dry/empty of buffer during the run.
It seems that you loaded prestained MW markers in precast gels so the problem is beyond sample or custom gel preparation. Moreover the bands are separated and focused which excludes problems with the sample. The lane-to-lane band pattern disturbance most likely shows inhomogeneous polymerization. Thus it is likely that the lot of the precast gels was bad. I suggest that you send the same image to the company you purchased the gels from and see what they will answer. Until that time, why not preparing the gels yourself?
Like George mentioned, since it seems that you used precast gels, the problem could be that 1) these particular lot of gels that you used were not good, 2) the running buffer formulation was not prepared correctly, or 3) that your gel box was not sealed correcltly and you had a small leak in the inner chamber .
I found out that it is the problem of the buffer. The pH of the buffer is lower than what it should be. I have made new buffers and the gel now runs perfectly fine. Thanks everyone .
Not sure about precast gels but for manually-cast ones, "unhappy" bands like this occur mostly due to buffer that has been re-used or that buffer has actually leaked out of the dam (between the plates)
might be this is because of used buffer and inadequate voltage distribution between electrode. i have also faced same problem but when it was operated in cold room with freshly prepared buffered figure out problem
- Badges
- Science method