Guidelines for Sample Submission

General Information

1. 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.

2. We recommend the use of coommasie blue stain over silver, but if you use silver stain make sure to use a mass spec-compatible silver stain protocol or kit.

3. Please provide an image of the gel and indicate the bands or spots that are to be analyzed.

4. 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.

5. Please provide a P.O. order form from your institute 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.

6. All P.O. numbers should be made payable to Helix Scientific Group, Inc..

7. Results will be sent via e-mail. All the data for each sample will be shown in an html link.

Background Information

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 (1-5ng). This is also very similar to the detection limit of proteins stained with silver and sypro ruby stains. Colloidal coommassie stains typically have detection limits between 10 and 20ng and coommassie 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. 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 coommassie stain. It is a good strategy if you are not sure if you will be able to detect your protein with coommassie 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 coommassie or if you will need to stain the remaining protein with silver.

Coommassie Stained Gels

1. Destain gel to a clear background so that bands can be easily seen.

2. If possible take a picture of the gel prior to excision of gel bands and submit photo (photocopy or electronically) along with the sample.

3. Excise gel band(s)/spot(s) with as little excess empty gel as possible.

4. Place the gel band(s)/spot(s) into a micro-centrifuge tube with some dd water.

5. Fill out Sample Submission Form.

6. Drop off or send samples and submission form to the core (Note: No need to send samples on dry ice).

Silver Stained Gels

1. It is strongly recommended that you use coommassie stain rather than silver.

2. If you can not visualize bands with coommassie try to scale up your isolation to increase the amount of protein until you get to coommassie detectable limits.

3. If you can not reach coommassie stainable levels you must use a mass spec-compatible silver stain protocol. Only use methanol and acetic acid during the fixing step. Do not use any solutions containing formaldehyde or glutaraldehyde to fix the gel. Refer to Shevchenko et al. (1996) Analytical Chemistry, 68:850-858 for more details.

4. It is better to have all the protein in one band/spot, but if you can not load more protein in one lane then run several lanes and combine the bands/spots.

5. Use 1.0 to 1.5mm gels.

6. Only stain the gel long enough (usually only a few minutes) to detect the bands of interest.

7. Take a picture of the gel and submit it (photocopy or electronic) along with samples.

8. Excise gel band(s)/spot(s) with as little excess empty gel as possible.

9. Place the gel band(s)/spot(s) into a micro centrifuge tube with some dd water.

10. Fill out Sample Submission Form.

11. Drop off or send samples and submission form to the core (Note: No need to send samples on dry ice).

Sypro Ruby Stained Gels

1. Sypro Ruby is a mass spec compatible protein stain with similar sensitivity as silver stain, therefore the same precautions (1-5 under Silver Stained Gels) apply to Sypro Ruby stain.

2. Since the stain is a florescent dye it can not be seen by eye. Please submit a picture of the gel and indicate the bands or spots to be analyzed.

3. Excise gel band(s)/spot(s) with as little excess empty gel as possible.

4. Place the gel band(s)/spot(s) into a micro centrifuge tube with some dd water.

5. Fill out Sample Submission Form.

6. Drop off or send samples and submission form to the core (Note: No need to send samples on dry ice).

Phosphorylation/Modifications

1. Samples for modification analysis must be coommassie stainable.

2. The more protein you can load in the gel and the more pure the protein is the better chance we will have of finding modification sites.

3. Send the sequence of the protein along with the sample. We will do a theoretical trypsin digestion to determine the peptide coverage. If peptide coverage is poor with trypsin another enzyme will need to be utilized.

4. Excise gel band(s)/spot(s) with as little excess empty gel as possible.

5. Place the gel band(s)/spot(s) into a micro centrifuge tube with some dd water.

6. Fill out Sample Submission Form.

7. Drop off or send samples and submission form to the core (Note: No need to send samples on dry ice).


Please send samples to:

Helix Scientific Group, Inc. 
1899 Mount Royal Drive, Atlanta, GA 30329