The use of gels for proteomics was quite common in early days, as easy means to remove contaminants from the sample, as well as means of sample fractionation before analysis.
Today we have the means to remove all but most persistent contaminants in solution, so please consult with us before performing the experiment (it may not be necessary to run the sample on a gel).
Gel preparation:
1. Gels should be low (4-12) % acrylamide. Higher percentages lead to reduced extraction efficiency.
2. If you need to resolve low MW components, it’s advisable not to use rather higher % Tris-Glycine gel. Tricine and Bis-Tris with MES buffer are excellent alternatives.
3. If you cast your own gels, please prepare them at least 24hrs in advance, and make sure to wash the well with Tris buffer before loading them with samples. This will prevent reaction of proteins with acrylamide monomers.
Gel visualization:
- Please contact us if you’re planning to use any other dye or imaging technique other than Commassie.
- Do not use covalently bound stains of any kind or use formaldehyde for staining without consulting us first. These stains change the proteins, add modifications and make it harder to extract the protein after digestion.
- “Stain-Free” by Bio-rad is a covalent stain!
- Do NOT use colloidal stains
- Commassie is both easy to destain and provides us important information. Faint bands are ~10ng, which is the lower limit of getting a good protein signal.
- Use of silver stain and other more sensitive stains might result in lower quality MS data. Consult us first to discuss!
Sample considerations:
1. If you reduce and alkylate the sample before running, please let us know.
2. Make sure not to overload the gel. Gels have maximum capacity of proteins both for the whole gel and specific bands (See here for a guide). Overloading the gels will result in contamination of nearby lanes, as well as altering migration times resulting is distortion of migration patterns.
3. Avoid sample precipitation in the well. This may happen due high sample concentration and/or rapid sample cooling after boiling. Precipitated sample will solubilize continuously during the run resulting the nullification of the separation.
Post-run:
1. Fix immediately! (this means add 5% acetic acid in methoanol or ethanol to precipitate the proteins in the gel. See this discussion on this topic). Proteins will begin to diffuse immediately after the current stops, with low MW protein diffusing faster than high MW proteins. If left for long, the proteins will diffuse into the buffer and coat the gel evenly.
2. As a rule of thumb, successful analyses require enough protein to stain lightly with coomassie stain (~10ng).
3. Please do not use colloidal stains as they are difficult to destain. Please do not use covalently binding stains, or formaldehyde based stains.
4. Please send us a picture of the gel before sending a sample!
5. Do not rely on Western blots as means of assessing sample suitability. While you might observe the desired protein, you cannot determine how pure your sample is.
6. If you do use Western blot to determine which bands to cut, please use the same gel you’ve used for transfer. The use of short transfer time, will result in 50-70% of the sample staying on the gel itself, which is enough for analysis.
7. If you decide to cut a different gel to the one you’ve run a Western blot on, you have to stain the gel. Different gels, even ones that ran in the same tank, will migrate slightly differently.
8. Cut bands should be placed in separate Eppendorf tubes without any buffer in 4C. Do Not Freeze!