Hi All,
I have been performing Reducing SDS PAGE for pure antibody where I could find additional bands apart from heavy and light chain with molecular weight around 75 to 100kDa and at 100 to 150kDa. I have been using DTT at a concentration of 50mM for reducing the sample. Kindly suggest me the probable reasons behind these additional bands in SDS-PAGE
Hi,
Purity is important to get only the expected band, so you need to double check the purity of your antibody.
Try to use equal volume of 4x loading buffer (protocol is shown below) which contains a higher concentration of DTT and boil the samples for 5 minutes at 96C.
200 mM Tris-Cl (pH 6.8)
8% SDS
0.4% bromophenol blue
40% glycerol
400 mM DTT
Add DTT to the buffer from a 1 M stock just before the buffer is used.
You can also load a lane with BSA as a standard lane so as to be sure about the quality of the gel and the running method.
If you still get the second band, it means that the second band is a product of the degradation of your antibody and to prevent this you need to use a potent protease inhibitor cocktail.
Hope it helps
Thanks for the reply Soran. Does low concentration of DTT lead to such multiple bands? These additional bands were observed only in silver stained gels and not in coomassie stained gels which is due to higher sensitivity with silver staining. Could you give me reasons if any , along with a documentary proof?
I wouldn’t say that low DTT concentration leads to multiple bands but a higher concentration of DTT helps to further denatures the protein by reducing disulfide bridges and thus helps to overcome some forms of tertiary protein folding which affect mobility.
Regarding silver and coomassie staining, if you don’t see the extra band on the overnight coomassie stained gels (Especially with InstantBlue stain if available), you don’t need to be worried about your result since a complete inhibition of protease activities is almost impossible. You can also carry out western blotting to confirm your SDS results.
Best
Thanks Soran. We are very much interested in silver stained gels and were eager to know what could lead to such additional bands. Do you have any reference which gives information about these additional bands? We didn't observe any additional bands in western Blotting.
Thanks for your time.
My suggestion for you is to leave 1 or 2 lanes blank and 1 or 2 lanes loaded with just loading buffer then load your samples in the other lanes. After staining three scenarios are possible as follow: First, if the contaminant band occur at 58kD and 64kD across the entire gel including the blank lanes, it means that you have keratin contamination which is a very common contamination in silver gels and in this case it happens during the preparation and loading of the buffers or the gel; Second, if you detect the contaminant band (58kD and 64kD) in all the lanes apart from the blank lanes, it means that you again have keratin contamination and comes from your loading buffer; Third, if you detect the contaminant band only in the lanes loaded with your product and with a different size than the aforementioned sizes, it means that the extra band comes from your protein and it might be contamination or degradation as we discussed it before.
To prevent keratin contamination, you need to wear gloves at all times during the preparation and loading steps. Also, use 0.45um nitrocellulose filter to filter all solutions.
Best
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