Director:
Belinda Willard, Ph.D.
Tel.: (216) 444-7170
Location: NN1-25
Fax: (216) 444-9404
Email: willarb@ccf.org

Support Personnel:
Dongmei Zhang, Ph.D., Lab Manager

Frequently asked questions

  1. How much protein do I need?

    2. Any band that can be detected with Coomassie staining contains enough protein for a successful analysis. In my experience, this requires approximately 1 pmol of protein, but the exact amount is dependent on the protein. The protein must be visualized to be cut from the gel.

  2. What about analysis of silver stained bands?

    3. The analysis of silver stained protein bands is certainly possible with mass spectrometric methods. The mass spectrometry is amply sensitive for the analysis of fmol amounts of protein so it is fundamentally able to sequence silver stained bands. However, two significant practical problems must be addressed and can destroy the effectiveness of the experiment. 1) Glutaraldehyde fixation modifies proteins in a manner that makes them impossible to digest. This reagent simply cannot be used in the staining procedure. 2) The background created by both protein and non-protein contaminants of the gel will mask the signals produced by peptides derived from the protein of interest. These contaminants are certainly also in Coomassie stained gels, but the inherently larger amount of analyte protein overcome the problems these species create. As a result, the ultimate success of experiments sequencing silver stained bands is generally dependent on absolute cleanliness and care at every stage of the experiment, including the sample preparation and electrophoresis step. It has been my experience that most laboratories find that it is easier and quicker to scale up to Coomassie blue-detectable amounts of protein than it is to identify and solve contamination problems.

  3. What is the turnaround time?

    4. Our goal is to be able to provide preliminary data within two weeks and a written report within three weeks.

  4. How pure does the sample need to be?

    5. Any protein that can be cut from a gel can be sequenced, no matter how many additional bands are present in the gel. There will always be some concern that a band is composed of more than one protein, but this should not affect the ability to detect and sequence peptides produced in the digestion.