Proteomics Core Services/ Guidelines

Services provided by the Proteomics Core Facility include:

1. Protein and peptide mass measurement
2. Protein identification
3. Protein quantitation: proteome-wide (SILAC or label-free) and targeted analysis (MRM)
4. Posttranslational modification analysis: phosphorylation, acetylation, etc.
5. Phosphopeptide affinity purification
6. De novo peptide sequencing

Proteomics Core Guidelines

Sample preparation for protein identification

1. Commercial mini-gel is good for protein separation with less chance of getting keratin contamination.

2. Stain the gel with Coomassie based staining. You can use a commercial kit or the following recipe.

• 3. De-stain the gel well (such as change buffer 3 times, each time wash 60 min)
• 4. Cut the gel band as small as possible. We cannot accept gel pieces that total in area more than 1 square cm per tube, otherwise it needs to be split into multiple tubes.
• 5. The gel band can be stored at 4ºC refrigerator for one week. For longer storage, stored in a minus 80ºC freezer.
• 6. If your protein band can not be visualized by Coomassie staining, Sypro Ruby staining is a good choice since it has similar sensitivity as silver staining but is cleaner. You need UV light to see the bands.

Recipe for Coomassie Brilliant Blue Staining (for mini-gels):

• 1. Fix gel in 100 ml 46 % methanol, 7 % acetic acid for 1 hour.
• 2. Stain gel in 100 ml 46 % methanol, 7 % acetic acid, 0.1% Coomassie Brilliant Blue R-250 (filter this before use) for 1 hour.
• 3. De-stain gel in100 ml 5% methanol, 7.5% acetic acid for 24 hours. Replace if needed.
• 4. Store the gel in 1 to 2% acetic acid in clean sealed sample tubes at 4 degrees C.

Sample preparation for protein quantitation

Please consult us first before starting protein quantitation project since each project can be quite different. Consultation ensures an appropriate workflow to be used.

To request Proteomics Services

1. Download the Excel sample form. Fill in a few things. Save it for future use.

2. Go to this link: http://www.med.upenn.edu/apps/my/itmat/proteomics

3. Type your PennKey and password. If you have an account to use, it will allow you to go further.

4. Select your account, add your e-mail address, confirm the e-mail and click Browse to upload the Mass Spectrometry form you just saved.

5. Click the Submit. Bring the samples to Room 852, BRB and follow the instruction on the desk next to the window.

If you have no account to choose, please download the instruction of SAM in the blue link under Place an Order, and forward the instruction to your BA who should be able to set up an account for you.

If there is any difficulty to set up an account, please call Jason Molli at 898-8264 or e-mail Jason: molli@exchange.upenn.edu