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Basic Guidelines and Protocols


Protocols

Protein Precipitation

FASP (Filter-Aided Sample Prep)

In-solution Tryptic Digestion

In-gel Tryptic Digestion

SDS-PAGE protein separation

SILAC media preparation

Basic Guidelines for Protein Sample Handling and Preparation

High purity preparation of a protein sample (or protein complex) is the key to successful mass spectrometry. It is very important to prevent or minimize contamination of your sample. Problem contaminants include keratins, polyethyleneglycol (PEG) and detergents.

Keratins

Keratin is a common protein contamination. It is extremely easy to contaminate your samples with keratins, which are ubiquitous and come from skin cells, hair, fingerprints, lint, lint from Kim-wipes, and dust in the lab. The smallest speck of dust or lint will most likely overwhelm your sample with keratin proteins. Great care is required to prevent keratins from showing up in MS analyses.

a.    Reduce the amount of exposure of the gel, gel equipment, and supplies to primary sources of keratins, such as skin, hair, clothing, etc. Reduce the amount of exposure to dust and particulates, both of which can be a rich source of keratins.

b.    Perform as much work as possible in a biological safety cabinet (BSC) or laminar flow hood.

c.    Thoroughly wipe down work surfaces with water and ethanol.

d.    Always use powder-free nitrile gloves. Don’t use Latex gloves. Latex is manufactured with proteins, which can contaminate your protein samples.

e.    Use of “low-binding” (but not silanized) polypropylene microcentrifuge tubes is recommended for sample storage and to prevent adsorptive losses of proteins and peptides.

f.     Use precast gels such as NuPage gels from Invitrogen and use their readymade buffers, loading dye, etc to minimize keratin contamination.

g.    Gel tanks are a common source of contaminants.  If your gel tank has been left to dry by the side of the sink for several days it is now contaminated with keratin from dust.

h.    When staining your gel, cover the staining tray on the shaker and wash the staining dish well before use.

i.   Communal lab chemicals (loading buffer, lab buffers, etc) are the most common sources of keratin. Do not use – make fresh.

 

Polyethyleneglycol (PEG)

PEG is strongly ionized and can drown out signal from the analyte of interest. PEG has a common repeat of 44 Da in the mass spectrum.

a. Avoid dish soap as it contains PEG. Either do not use or rinse glassware with very hot water followed by an organic solvent rinse.

b. Use only HPLC grade solvents.

c. Do not store organic solvents in plastic tubes. PEG contaminants can leach out of plastic tubes.

d. We recommend Eppendorf brand microcentrifuge tubes.

Detergents

For in-gel submissions, most of the detergents (<0.1% SDS, etc) can be cleared from the system before mass spectrometry analysis. Avoid using Triton-X100, SDSAPS, Tween 20, NP-40, etc, which can adversely affect results. If you need to clean up your sample, you can use detergent spin columns, gel filtration, dialysis, etc. to remove the excess detergents or use the Filter Aided Sample Preparation (FASP) method for sample preparation. Please utilize one of the mass spec friendly detergents listed below:

Mass Spec Friendly Detergents:

1)N-octyl-β-glucopyranoside

2)ProteaseMax

3)Progenta™ Acid Labile Surfactants

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