I have a purified enzyme which is giving good activity on its substrate but I am not able to visualise any bands on SDS-PAGE gels. How much is the minimum of protein am I need to load?
The minimum maount of protein to load varies greatly depending on the gel staining method. Different Coomassie stains have different sensitivities (e.g. 100 ng for Coomassie Brilliant Blue R-250, about 30 ng for Colloidal Coomassie Brilliant Blue G-250). Silver is more sensitive and can detect as little as 5-10 ng.
The minimum maount of protein to load varies greatly depending on the gel staining method. Different Coomassie stains have different sensitivities (e.g. 100 ng for Coomassie Brilliant Blue R-250, about 30 ng for Colloidal Coomassie Brilliant Blue G-250). Silver is more sensitive and can detect as little as 5-10 ng.
Hey Ravinder,
in a coomassie stain G250 the detection limit for proteins is around 30 ng, if you use silver stain you can go down to 1 ng.
See also the publication attached to this answer.
All the best,
Janko
Article Highly Sensitive and Fast Protein Detection with Coomassie B...
If your protein amount is more the 10 ng coomassie stain G250 can be used for detection but if it is lower the that then silver stain will be used.
Thankyou all...
I have determined concentration of my protein but it is so low that the value doesnt fit between 1-10 ug/ml (Using Bradford standard curve/Sigma). Is there any other more sensitive method to know its concentration so that i can load accordingly to get obsevable bands. I have even tried silver staining but no observable bands. Any sugesstions????
Dear Ravinder
Bradford is the sensitive method and you need no other protein measurment procedure.
You must concentrate your samples or precipitate your proteins to elevate its availability and then run a PAGE.
Masood has rightly quoted.... You need to concentrate your protein sample... There are 10-50KD centrifugal devices available by Millipore and Pall starting with low capacity of 2 ml to 50 ml ... Use these centrifugal tubes or go for chilled acetone precipitation. The beauty of the acetone treatment is that the activity of the protein is never lost... after acetone precipitation under chilled conditions you can recover the precipitate by centrifugation and resolubilise your protein in the buffer system you desire for may be sample buffer and then run an SDS-PAGE.... I am pretty sure silver staining will definitely work then if not Coomassie Blue... Gud luck....
Yes, I have done acetone treatment after 30 KDa millipore concentrator and did SDS-PAGE . I was able to see bands using silver staining but still very faint. Anyway, I think purifying more protein is needed.
Thanks everyone.
Jankos method is good but you can circumvent Methanol usage with this protocol...
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149902/
Depends how much volume of 0.07mg/ml protein you have Subhomoi. You can concentrate 30-50 times in a millipore concentrator, thats upwards of 2 mg/ml. Say, if you have 15 ml of your protein, you can bring it down to 250ul. Use the concentrator with right cut off size.
As some one already suggested, silver staining is more sensitive than Coomassie blue G-240 staining. It is also possible to re-stain Coomasie blue stain gel by silver staining. So if I were you, I would first stain the gel with Coomassie blue and, if unsuccessful, then with silver staining.
I want to know that whether SDS-gel can be restained with Coomassie G-250 staining solution if the bands didn't came in silver staining?
Niyam, changes are that if you have not gained staining by silver, you are pretty unlikely to get a better answer by Coomassie - the sensitivity of silver is higher than that of CBB G-250 (see Elsa Lauwers' answer above). Depending on what you have done to your sample you may have lost some protein (plastics, membrane filters and general processing steps will lead to losses). Hope that you are able to find your protein.
hi , i got the same problem. i get a good number of virus particles after TEM observation. but on running SDS-PAGE gel i could not get any band. i have used nano drop to check concentration of protein 280. I got the concentration 0.328 mg/ml in 2ul .i have 200 ul total sample. how could i concentrate it and get results on sds-page?
CBB binds to basic amino acids. So it varies from protein to protein. However, 100 ng should be easily visible with CBB R-250. In case if your protein is not abundant, go for colloidal CBB staining (which can visualise as little as 20 ng) or silver staining if you want to just see the band. All the best.
Sensitivity of staining depends on the protein, as different proteins interact differently with CBB. It also depends on the gel thickness and many other parameters.
If sensitivity is a problem, you can improve it by staining with RuBPS, a fluorescent dye originally proposed by Rabilloud et al. (doi:10.1002/1615-9861(200104)1:53.0.CO;2-C). It is easily prepared and quite cheap (or available from Sigma, but not cheap). I have published a detailed protocol in doi:10.1007/978-1-59745-542-8\_14
100 ng protein for CBB R-250, 30-40 ng for G-250 and 5-10 ng for Silver staining.
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