Staining & Drying
Coomassie blue: MDS uses Coomassie brilliant blue R250 as our standard O'Farrell staining procedure. This method involves an alcohol-acetic acid fix, acetic acid rehydration, and an acetic acid destain. Generally, a load of 1 ug of purified protein gives a highly visible spot on a 2-D gel. Although not as sensitive as silver staining, it gives quantifiable results, because the linear range for plots of stain density versus ng protein is broad and the method is reproducible. For quantification, we use Coomassie blue calibration strips containing known amounts of bovine serum albumin per mm2. These strips are stained and destained with the gels and are used to determine ug of protein in each spot based on BSA equivalents.
Coomassie blue calibration strip: The calibration strips accompany the slab gels through gel staining, destaining and drying. The calibration strips consist of serial dilutions of bovine serum albumin polymerized in 9 layers of 10% acrylamide. Each layer of the strip is approximately 1 cm x 1 cm x 0.75 mm. Exact measurements of mg BSA/mm2 for each layer have been made. The strips are used to generate a BSA standard curve showing the relationship between mg protein and stain darkness. Thus when calibration strips are used, the integrated density within a spot or band outline may be automatically converted to mg protein relative to BSA.
Silver staining: Our silver stain is based on the ammoniacal silver/formaldehyde method of Oakley. This method involves preliminary glutaraldehyde treatment of the slab gel to fix proteins by crosslinking. The pre-treatment also adds glutaraldehyde side chains to the proteins, increasing sensitivity since these groups are sites for silver deposition. We find this method to be 10-100 times more sensitive than Coomassie blue staining, depending on the protein. Generally, a load of 40 ng of purified protein gives a highly visible spot on a 2-D gel. However, some proteins detectable by Coomassie blue staining don't stain at all with silver. Although silver staining is very sensitive, this method gives qualitative results rather than quantitative results. This is because the linear range for plots of stain density versus ng protein varies widely from protein to protein. In addition, day-to-day variability in stain shading and background intensity are observed.
Carbohydrate staining: For this stain adjacent hydroxyl groups of sugars on glycoproteins are oxidized to aldehydes and subsequently labeled with a hapten. The final color reaction is achieved by Western blotting. Sensitivity varies with proteins and ranges from 100 ng to 500 ng.
Deglycosylation of proteins: Procedures for deglycosylation of proteins will be optimized and standardized using Biorad protocols. Enzymes in the Biorad kit provide for cleavage of Asn-linked as well as Ser/Thr linked oligosaccharides. Gel mobility shifts indicate whether deglycosylation has occurred.
Transparent drying: All stained gels are air dried between cellophane sheets unless otherwise requested. Although this treatment adds a day to the turnaround time, the advantages of easier storage, little or no curling, good color retention, overlay capability, scanning capability, and overhead projection capability offset the time delay. Since there is quenching by the cellophane, gels scheduled for autoradiography and fluorography are not transparency dried.
Paper drying: This is an optional drying method for stained gels. All enhance-treated slab gels scheduled for fluorography as well as unstained gels scheduled for autoradiography are dried onto thick filter paper unless otherwise requested.
Electronic Documentation: Photograph of gel for notebook records.
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