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Enzymatic/Using Cleavage of Proteins

The majority of samples submitted for protein identification/internal protein sequencing are submitted as single, Coomassie Blue stained gel bands or spots that are then subjected by the MRF to ingel trypsin digestion. The service charge for in gel digestion includes carrying out a control digest on an otherwise identical section of gel (supplied by the investigator) that does not contain protein. The purpose of this control is to enable rapid identification of reagent and trypsin autolysis products. Upon request, a variety of other enzymes (e.g., lysyl endopeptidase, chymotrypsin, Staphylococcal protease, etc.) may be used in place of trypsin. Although the MRF will also carry out enzymatic digestions on samples that have been blotted onto PVDF, the MRF strongly encourages investigators to avoid this unnecessary step and instead, to prepare these samples as described and to submit them as Coomassie Blue (either R250, G250 or "colloidal") stained gel bands. Although proteins that have been stained with silver (e.g., Electrophoresis 18, 349-359 (1997)) may be digested, our experience is they do not digest as well as proteins that have been stained with Coomassie Blue. The sensitivity of detection with "colloidal blue" appears to be about 25 ng. Samples that do not require SDS PAGE purification may be submitted dry (after vacuum centrifugation in a Speedvac) in 1.5 ml Eppendorf tubes that do not contain O-rings. Although these samples generally may contain up to the equivalent of 50 µl of 0.5 M non-volatile salts, the investigator should email the details of these samples (i.e., exact buffer composition and estimated protein concentration) to the Protein Chemistry Section prior to final sample preparation.


	Enzymatic/Using Cleavage of Proteins The majority of samples submitted for protein identification/internal protein sequencing are submitted as single, Coomassie Blue stained gel bands or spots that are then subjected by the MRF to ingel trypsin digestion. The service charge for in gel digestion includes carrying out a control digest on an otherwise identical section of gel (supplied by the investigator) that does not contain protein. The purpose of this control is to enable rapid identification of reagent and trypsin autolysis products. Upon request, a variety of other enzymes (e.g., lysyl endopeptidase, chymotrypsin, Staphylococcal protease, etc.) may be used in place of trypsin. Although the MRF will also carry out enzymatic digestions on samples that have been blotted onto PVDF, the MRF strongly encourages investigators to avoid this unnecessary step and instead, to prepare these samples as described and to submit them as Coomassie Blue (either R250, G250 or "colloidal") stained gel bands. Although proteins that have been stained with silver (e.g., Electrophoresis 18, 349-359 (1997)) may be digested, our experience is they do not digest as well as proteins that have been stained with Coomassie Blue. The sensitivity of detection with "colloidal blue" appears to be about 25 ng. Samples that do not require SDS PAGE purification may be submitted dry (after vacuum centrifugation in a Speedvac) in 1.5 ml Eppendorf tubes that do not contain O-rings. Although these samples generally may contain up to the equivalent of 50 µl of 0.5 M non-volatile salts, the investigator should email the details of these samples (i.e., exact buffer composition and estimated protein concentration) to the Protein Chemistry Section prior to final sample preparation.