2-D Gel Electrophoresis
Routine 2-D Gel Service: Routine service includes sample preparation (solubilization), running both dimensions, staining of gels and presenting stained gels and digital images on a disk.
Accepted Sample Submissions Include:- Frozen cell pellets or tissues
- Cell or tissue lysates
- Purified proteins
- Serum - plasma - urine
- Samples are generally prepared using urea, CHAPS, carrier ampholytes and DTT.
- Pre-fractionation of samples is usually required for 2-D electrophoresis. Specimens can be selected on the basis of solubility (using buffers of increasing stringency), charge (ion exchange chromatography) and/or molecular weight (micro filtration or size exclusion chromatography).
- High abundance proteins including albumins and immunoglobulins are removed from serum and plasma prior to electrophoresis.
- Selection of proteins can also be achieved by studying individual cellular compartments (organelles) purified by differential centrifugation
Please Note: Sample fractionations are not included in Routine 2-D Service. The costs associated with these procedures are estimated on a case-by-case basis.
2-D Electrophoresis: Following sample preparation, the IEF dimension is performed using 7 cm or 18 cm IPG strips of selected pH range (3-10, 4-7, 3-6, 5-8, 7-10, 6-9, 3-7, 6-11, 3.5-4.5, 4-5, 4.5- 5.5, 5- 6, 5.5- 6.7).
Molecular weight standards are incorporated at the start of the second dimension.
SDS-PAGE for 18 cm strips is performed using pre-cast gradient or fixed percentage, 22 x 22 cm format tricine or tris-glycine gels.
Depending on the protein(s) of interest, 20 % gels are used to resolve small proteins, 12 % gels to resolve mid-range proteins, and 7.5% gels for large proteins. SDS-PAGE for 7 cm strips is performed using Novex NuPAGE 4-12% Bis-Tris ZOOM gels.
Staining of Gels and Acquisition of Digital Images
Three methods are available:- Regular Silver Staining (w/ glutaraldehyde): sensitivity > 10 ng
- Non- Destructive Silver Staining (glutaraldehyde omitted): sensitivity > 50 ng
- Fluorescence labeling with SYPRORUBYT: sensitivity > 10 ng