I have stained my SDS-PAGE with coomassie blue but bands are very light. Probably, the stain was not good. Can the gel be stained again with new coomassie or silver stain?
Will the staining be efficient?
Yes, You can restain the gels. Its upto you, If you wants stain with coommassie then can use by freshly prepared and if you think that protein concentration is low, can try silver staining. But you have to destain complete gels before silver stain otherwise you will get poor staining or much background. I did this thing many times and it works.
Yes, You can restain the gels. Its upto you, If you wants stain with coommassie then can use by freshly prepared and if you think that protein concentration is low, can try silver staining. But you have to destain complete gels before silver stain otherwise you will get poor staining or much background. I did this thing many times and it works.
Hi Ravinder
the first thing to think about it is the staining of the marker bands not the sample bands because may be the your samples contain little amount of protein which appears as a faint band
Yes, you can restain coommassie gels. Just wash the gel after the destain to remove any destain remaining and put the gel back into coommassie stain. Then destain as usual. If you destain overnight, you can also decrease the time in destain so that your bands are still dark when the background has destained sufficiently. You can also restain with GelCode Blue if you are using that instead of a traditional coommassie stain.
Bands are faint because the protein loaded are very few. Did you check if your protein determination contains the right blank controls? Triton in the extraction buffer react a lot with Bradford.
Remember, the rigth control are water and extraction buffer, than you can check you protein.
If I am right 10 ul of 1% triton give a 0.3 absorbance against water in a 5ml bradford. Better use 5 ul that give almost nothing
You can stain and destain with coomassie as many times as you wish. I give you a modified recipe for coomassie staining that in my experience is particularly efficient for slight bands: 0.25% w/v coomassie, 30% v/v isopropanol, 20% v/v methanol. Stain for 10-15 min, then destain with 10% v/v CH3COOH. You will have the complete removal of blue background after an overnight incubation in 10% v/v CH3COOH. If you cannot see any band with this staining try with silver.
To add to the previous excellent suggestions:
1. Destaining can be sped up if you use hot methanol/acetic acid (and I mean almost boiling!). The lab will smell like a fish-and-chip shop for a while, but you get clear background very quickly.
2. Silver staining over Coomassie-stain can work, but is usually quite ugly. If you can see your bands (even faintly) on Coomassie, I would run a separate gel with 10-30% sample and silver stain from fresh - it is about 10-100x more sensitive. Beware, though - some proteins stain negatively on silver stain (e.g. BSA!!). I find that the pattern is much crisper if the gel was not Coomassie-stained first.
Good luck!.
My experience is similar to Anna's. I once stained a gel with Coomassie but did not like the result and fixed it in methanol/acetic acid for silver staining. Well... it worked but in a weird way: most bands were stained negatively. Very beautiful (!) but hardly useful if you want a nice image.
Hi Ravinder,
My experience is also similar as other contributors. I destained and restained gels with Coommasssie (2.5%) with no problem at all. But coommmasie destained gels never stained well with silver stain. Either I had too high background or high background with negatively stained bands (as Alexander mentioned).
Silver staining method has higher sensitivity and the bands which usually do not stain well in coommassie can be seen by this method. The problem is the bands which appears well in coommassie (considering higher abundance of protein) appears negatively stained with no color.
I would suggest running a fresh gel for silver staining.
Good luck!!!
You can Coomassie stain (R250 detection limit ~25ng) your gel as many time you want. For Silver stain (10-100 times more sensitive than R250) the Coomassie stained gel may not be as clean as a new gel. So, if your sample is not precious I would recommend to re-run your gel and then go for silver stain. BTW I agree with Jesus and Anna.
Try this colloidal Coomassie staining with g-250 it is nearly as sensitive as silver staining but you 1) cannot overdevelop as for silver staining 2) only 3 steps necessary and 3) no need for toxic methanol and smelling acetic acid http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149902/
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