Hello everyone,
Hope all are fine. I have a doubt about SDS-PAGE. I have done 4 gels (details were given in the attachment file) for the same known targeted protein contains two bands. In all the cases, I have seen only one band and another band is not clearly visible.
Can anyone help me out to sort this problem?
Note:
1) Acrylamide mixture was freshly prepared
2) pH was also checked for each and every gel preparation
Thank You in advance!!!
What are the sizes of the two bands that you are expecting ? And what are you showing in these gels ? I mean, what does each lane represent ?
Dear Srichandrasekar Indhuprakash
What was the protein concentration and was the concentration of all run samples the same?
If your sample was contaminated, the absorbance reading of it corresponds to the protein + contaminant concentration, not just the protein.
What sizes of bands are you looking at? Can you explain what is loaded to each lane?
Respected Mehrdad Mohebi Shahpour Abadi Merve Oztug Praveesh Valissery
One of my protein sizes is 66kDa and the other one is 28kDa in the same kind of several batch samples. I have loaded 100 microgram concentrations for all the wells. All the mentioned lanes are the same sample. The first lane in all gels is a protein ladder and other lanes are unknown proteins. The concentration was calculated with the help of UV spec
The proteins in each gel look different from the other. It is hard to belive that they are from the same sample batch at the same concentration; unless some sort of proteolytic degradation has ruined your sample between each run. But that is unlikely as there is no pattern here. It is hard to infer much from these gels.
You can improve these gels by following the comments mentioned in the attached image.
Hi Srichandrasekar Indhuprakash
Our lab makes use of Bio-Rad PAGE Setup and with the standard components we are able to produce replicable results. From the gels uploaded, it is clear that they require longer staining time (5 hours or overnight) and destaining (3-4 hours or longer depending on the destaining). Make sure that after destaining the background of the gel is clear so as to enable easy visualization. All the images show lot of variation in terms of staining, destaining, protein quantity added and run time. Try sticking to one make viz., Bio-Rad or Medox and then standardize.
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