I am running an insoluble protein in SDS gel to check its size.
I prepared 25mg/mL stock solution and add 1-10 uL of it to my loading buffer but I cannot get the image.
Does anyone know how much proteins are usually added to each loading buffer in a lane?
Hello, Huihun Jung
If you use Coumassie staining, you can detect ut to 100ng of individual protein per line (on mini-gel). 25 mkg is 10-100 fold more, then needed. Check 2 points:
1) The sample buffer is fresh (2-mercaptoethanol does not evaporate). Proteins may be S-S crosslinked.
2) The time of sample boiling is enough.
You can staining the gel with it's upper part. May be, proteins do not introduce in the gel and remained in the well.
Hi Huihun Jung
It depends on the thickness of your gel, and the method of staining. Normally for a gel of 1mm thickness, you can load about 20 microgram of protein if you stain it with C. Blue. with the silver staining method, you can detect around 1 miro gr easily.
Before that, you must be sure that your protein assay method for protein concentration determination is compatible with the components including in the extraction buffer. If it is not compatible for example with SDS, DTT, or other materials in the solution you might have overestimated the protein concentration. In fact it has been very low amount of protein in the sample and that is why you have not been able to detect it. I recommend that you measure the protein content of your sample by at least two dififferent method before loading to the gel.
If your protein determination in your stock solution is correct at least with two different methods you need to check if you have any very high MW bands (or smears) which might even not getting into the gel (remaining in the loading pocket or at the boundary between the stacking and separation gel, especially if your protein is not soluble even in SDS). That's why I always stain all my gels including the stacking gels with all loading pockets, even with commercial gels. If you don't see any high MW bands including loading pockets, you might have a problem with the SDS-PAGE itself (SDS-Loading Buffer, SDS-Running Buffer, the buffers of the gel itself, if you made it by yourself). Just make sure that the MW markers are separated as they are supposed to, and all the buffers and parameters above are correct.
try to increase the injected volume to 20 uL and you will get good results.
