First time users, non-affiliated researchers, those interested in protein modifications and anyone wanting to submit large numbers of samples at once (more than 15) are urged to contact the facility prior to sample submission. We recommend the use of coomasie blue stain over silver, but if you use silver stain make sure to use a mass spec compatible silver stain protocol or kit. Please provide an image of the gel and indicate the bands or spots that are to be analyzed. Please fill out the Sample Submission Form and answer the questions listed. The more information we have about the nature of the samples the better we will be able to serve you. Please provide a Harvard 33 digit billing number or a P.O. order form containing a P.O. number, the amount of money on the P.O. number, and the billing address for sending the invoice. Samples will not be analyzed unless your P.O. form is received. All P.O. numbers should be made payable to Harvard University. Results will be sent via e-mail. All the data for each sample will be contained in an html link. For the fastest turnaround time please submit samples on Monday. We only process one set of sampes each week starting on Monday. Turn around times vary between one to two weeks depending on our backlog.
The detection limit of proteins from SDS-PAGE gel bands through the use of LC/MS/MS is in the femtomole amounts or on a weight basis in the low nanogram range. This is also very similar to the detection limit of proteins stained with silver and sypro ruby stains. Colloidal coomassie stains typically have detection limits between 10ng and 20ng and coomassie brilliant blue has a detection limit near 50ng. The detection limits of these stains will be dependent on many variables such as the thickness of the gel and the width of the lanes. While the detection limit of protein staining is on a weight basis the detection limit of protein with the mass spectrometer is on a molar basis, therefore the higher the molecular weight, at the same mass, the higher the detection limit will be for the mass spectrometer. For example 1.0ng of a 20kd protein is 50fm, while 1.0ng of a 200kd protein is only 5fm. Both proteins will have similar stain intensities, but there is 10 times less protein on a molar basis from the 200kd protein. In general the detection limit for protiens from gels will be 2-5ng for proteins less than 75Kd, 5-15ng for protein between 75-150Kd, and 15-25ng for proteins from 150-250ng. Protein stains also detect total protein, while the mass spectrometer detects proteins individually. It is very common for gel bands to contain several and sometimes dozens of proteins.These proteins will result in a band on the gel that is detectable with silver stain (i.e. a total of 5.0ng of protein), but there may not be any single protein in the band that would be detectable with the stain by itself and therefore most likely not detectable by the mass spectrometer. For these reasons we encourage the use of coomassie stain. It is a good strategy if you are not sure if you will be able to detect your protein with coomassie to run a small amount of your sample (1-5%) on a gel and use silver stain. In one of the lanes of the gel you can run a known amount of protein. We would suggest running 5.0 to 10.0ng Phosphorylase B (Sigma-Aldrich P4649) or B-Galactosidase (Sigma-Aldrich G8511), or another protein that you have available. The stain intensity of your sample and the known proteins should indicate whether you will be able to detect the rest of the protein with coomassie or if you will need to stain the remaining protein with silver.