Norms for Sample Submission

Table 1: Mass dependence of protein concentration for mass spectrometric analysis of soluble protein*

Mass (Da)

Concentration required in mg/ml
(20-50 µM)

500

0.01-0.02

1,000

0.02-0.05

2,500

0.05-0.13

5,000

0.10-0.25

10,000

0.20-0.50

20,000

0.40-1.00

30,000

0.60-1.50

40,000

0.80-2.00

50,000

1.00-2.50

60,000

1.20-3.00

70,000

1.40-3.50

80,000

1.60-4.00

90,000

1.80-4.50

100,000

2.00-5.00

* This is a general guideline; current instrumentation is more sensitive.

Before submitting samples for analysis all potential users of the facility are encouraged to discuss their particular needs with the personnel of the facility.

Sample size:

The following recommendations are general guidelines. Our current instrumentation is extremely sensitive and most likely will be able to perform a good measurement with less material that indicated.

Soluble samples: For gel-free protein characterization, we find that 5-10 µg of total protein will provide enough sample for several good LC-MS analysis. This will allow us to confirm identifications and use exclusion lists to identify some of the lower abundant proteins in the sample.  The concentration (mg/ml) of the sample needed depends on the molecular mass of the protein/peptide.  Table 1 is given for reference purposes.

PAGE bands: Coomasie-stained gels are preferred. Users are encouraged to scale up sample preparation to this level of detection; it is our experience that spending some time doing method development will result in quicker, less ambiguous protein identification. If not possible, syproruby stained gels can also be submitted.

The amount of sample required to ensure an adequate protein identification attempt is approximately 50-150 fmol  (approximately 3-15 ng of a 100 Kd protein) and represents the lower limit of detection in the average colloidal Coomassie blue stained mini-gel. Silver stained gels, even MS-compatible silver stains, are strongly discouraged. Silver staining protocols differ widely in their detection limits and differences can even occur between gels stained with the same reagent/protocol combination. This is chiefly due to the subjective developing step. The silver deposition on the gel surface can also affect the peptide yield following protein digestion. We have success in identifying proteins in silver stained gels that are present at a level of greater than 200 fmol, but keep in mind that it is difficult to quantify protein levels using these methods, even with standards present on the gel.

Radioactive Sample Permissible Levels. None.

Please review the following document: keratin-free environment

Last modified: Mar 26, 2013
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