Amersham 2DE Training

**Note this page is material from John Tullai and if you have a link problem try here

Miscellaneous Q & A From the Sessions:

In this section, the answers are either placed in the body of this page, or, where applicable, a link to download a file (usually a pdf) is provided.

  • Ruby versus Cy 2,3,5 crosstalk question:

The excitation and emission filters are distinct for all the dyes and Sypro ruby. The major problem is that Sypro ruby fluoresces across such a wide range (excite around 400, emission around 630) that you will get some Sypro ruby signal if you were to try to re-image the Cy dyes. This means that once you’ve stained with Sypro ruby, you can no longer go back and re-acquire a Cy dye image, since the quantitation would no longer be correct. So there is cross-talk in the Cy dye channels due to signal coming from Sypro ruby.

For low salt digestion, use 7-20mM ammonium bicarbonate/50% methanol instead of 50mM ammonium bicarbonate/50% methanol solution.

Cy Dye bound to the peptides and measurement with Mass Spec:

The bottom line is that due to the very low abundance of the Cy Dye to be potentially bound to the protein that it is not an issue in detection with the minimal labeling (to the lysine residues) (the saturation labeling through the cysteine residue is mass spec. compatible and needs the Cy Dye MWt to be added into the data base search for being bound to the cysteine residues)

Immobilized pH gradients-table of gradients. Download the pdf (1 MB)

Sypro Ruby staining protocol of 2D gels. Download the pdf (68K)

The reversible stain for storing IPG strips at room temp. Download the pdf (12K)

Bind-silane method for gelbond-backing gels. Download the pdf (8K)

Reference describing the method to make new Sypro Ruby stain. Pubmed Reference.

Reference for running IEF of proteins with basic pI. Pubmed Reference

Basic pH gradient with high sample loads. Pubmed Reference. Brief method pdf (4K)

Reference describing increase in [DTT] to 3% to improve basic pI resolution. Pubmed Reference.

Acid violet staining of immmobiline gels and strips: Patesotos et al. (1988) Electrophoresis 9:488-496. Download the pdf of the protocol (4K).

The use of glycerol and isopropanol in the rehydration buffer and discussion of electroendosmosis. Download the pdf

Test sample protocols-trial preps that almost always work well for 2DE:

  • Carrot Seed (this is the one we did in the class) (pdf 16K)

The use of IPG strip with hydrophobic proteins. Pubmed Reference.

Capacity of polyacrylamide gels. Pubmed Reference.

Diffusion Blot protocol-western method used for backed gels-gels can then be further processed (Spot picker, etc.). Download the pdf (4K).

Spot-picker parameters for unbacked gels. Download the pdf (4K).

The Tris tricine buffer system for the DALT 12. Download the pdf (4K)

Molecular Wt. versus gel percentage – Look at page 635 of the Amersham 2002 catalog-the table is at the bottom

The kits for membrane bound protein extraction etc is here: http://www.genotech.com/

Cell Wash for cultured cells prior to harvesting for IEF:

Salt carry-over from growth medium or wash solution can be significant. Salt-free buffer/osmoticum should be used for washing (10 mM Tris / 250 mM sucrose pH 7.0)