This depends on the complexity of the sample that was loaded on the gel. From our experience we have identified as many as 70 proteins from a single gel band when complex cell lysate was ran on 12%T minigel. We strongly recommend not to overload the gel as it will affect the resolution and separation of proteins. Moreover, it can result in “carry-over” of abundant peptides in LC resulting in identification of same protein repeatedly in different samples.
Yes, if it is stained by mass spectrometry-compatible silver stain protocol. Many commercial vendors sell such kits.
Negative staining techniques such as zinc stain are compatible with mass spectrometry. Many of the fluorescent stains are also compatible with mass spectrometry.
If sample is to be “in-solution digested,” we can use any one or combination of commercially available sequencing-grade proteolytic enzymes. For “in-gel digestion", the repertoire of the proteolytic enzymes becomes slightly more limited. Note that many high molecular weight enzymes are not as efficient for in-gel digestion as trypsin and chymotrypsin.
Yes. The simplest and cheapest way is to use precipitation technique like TCA and acetone precipitation. The downside of these methods is that once precipitated, some proteins are hard to resuspend in the buffer of choice. At Northwestern Proteomics, we offer a service to remove the detergent using Pierce detergent removal kit. We have successfully used this kit for removal of octyl maltosides and SDS from the sample. It should be noted that success of all any technique will depend on concentration and type of the detergent.
I have my protein complex in a buffer with very high salt concentration plus 5% glycerol. Is this buffer compatible with mass spectrometry?
No. Salts and glycerol are not compatible with mass spectrometry. If the samples contain salts or glycerol, we will desalt the sample either by using a zip tip or C18 spin columns.
You can use in house made stain. But note that presence of acetic acid and methanol can modify proteins. We recommend to use commercially available Coomassie blue stains as they are solvent free and are more sensitive than conventional staining. We do not have any preferred commercial vendors.