I am using 4-20% biorad precast mini gel and protein ladder is also from biorad. I have attached the image of membrane. Running voltage- 100 V for 1 hr at room temperature and 70 V for 2.30 hr at 4C. I am stuck with the crooked ladder. Lower bands of ladder become wider and it happens twice back to back. Please share the suggestion if you had the same experience or know how to correct it.
Thanks
Hi,
You see problems with western blot could arise due to several reasons, you have to hegate them one by one.
Since you are using a pre cast gel, there is very little to no chances of something wrong with the composition of the gel.
Secondly, you must check if the buffer you are using is freshly prepared and is of the correct composition.
For the running of the gel the protocol you are following must have definitely be standardised, however, we would run the gel at 60-70V for the first 1.30 hrs and then increase to 100 for the rest of the time. (I strongly feel it has to do something with the conditions of running the gel, so please check the preferable voltage of running your gel)
Now coming to transfer, though it doesnt seem to be an error of transfer, check the buffers and the conditions of transfer.
All the best.
If everything else is okay and the marker and the nearby lane is the issue, it is mostly due to different salt concentrations in your samples and the ladder. Maybe you are loading some high-salt extracts? You can try leaving a lane, between the marker and samples. If there is nothing unusual about the sample prep (simple lysate with loading dye), try adding 1:1 loading dye in the ladder lane too.
Best, Manoj.
Hi Anita,
seems to me that you might have an issue with some high-abundance proteins in your samples (with a molecular weight of approx. 60 to 70kDa like, e.g., albumin?).
What size is your protein of interest? If it is
Thank a lot for your suggestions, Suraiya and Manoj.
I am using all ready to use buffers from Biorad and using freshly prepared too (Ph checked). So i dont think that is the problem.
I surely try diluting my ladder with loading dye and keeping one lane gap in between. That seems right to balance salt conc. difference.
Thanks again for valuable inputs.
Regards
Anita
Hi Christian,
Thanks for explanation and suggestions.
I am using mouse serum for IgG and IgM. My protein of interest- 75, 50 and 25 kd (light and heavy chains of igs) I tried dilution (1/10 and more) but did not get publication quality image. So this time i tried BCA and used 20 ug/ lane protein for western and got this type of ladder...and bands were also not looking good.
I will try size exclusion spin columns this time.
Thanks for your help.
Regards
Anita
As far as I concerned, you have to monitor the level of running buffer that is present between the middle chamber. If the running buffer drops during the running, there is cent per cent of chances of getting this kind of band. The upper portion of the gel is perfect because there is a full of running buffer during the start of the running, and as going to downwards there must be depletion of the buffer that makes this kind of structure to the lower section of the gel.
This also depends upon the western blot apparatus. You must have to set up your experiment for getting better results.
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