Tricine/Polyacrylamide Gel Electrophoresis

Used for pilin processing analysis but generally useful for resolution of small (15-35 Kdal) proteins of similar size.

See
Strom, M. S., D. N. Nunn, and S. Lory. 1993. A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proc. Natl. Acad. Sci., 90:2404-2408.
or
Strom, M. S., and S. Lory. 1992. Kinetics and sequence specificity of processing of prepilin by PilD, the Type IV leader peptidase of Pseudomonas aeruginosa. J. Bacteriol. 174:7345-7351.
for all the references.

Reagents:

• Anode Buffer (+): 200 mM Tris pH 8.9 (can dilute from 10X stock)

• Cathode Buffer (-): 100 mM Tris/100 mM Tricine/0.1% SDS (no need to pH, but will be ~8.25) these can be made as 10X stocks and diluted before use

• Gel Buffer: 3.0 M Tris, pH 8.45/0.3% SDS

Note: the pH's given for the anode and gel buffers are essential

• Stacking acrylamide: 48% acrylamide/1.5% bis-acrylamide

• Separating acrylamide: 46.5% acrylamide/1.5% bis-acrylamide

• Sample buffer: (add equal volume to sample), for 20 ml:

5 ml 0.5 M Tris, pH 6.8

4 ml 20% SDS

1 ml 2-mercaptoethanol

4 ml 50% glycerol

0.004 g bromophenol blue

6 ml water

Pouring Gels

For each minigel (Hoeffer "Mighty Small" or BioRad "Mini Protean-II"--scale up as required):

• 1. Separating gel:

  15% gel

  2 ml separating acrylamide

  2 ml gel buffer

  2 ml 50% glycerol

  10% gel

  1.22 ml separating acrylamide

  2 ml gel buffer

  2 ml 50% glycerol

  0.78 ml water

• 2. Stacking gel:

0.25 ml stacking acrylamide

0.75 ml gel buffer

2.0 ml water

20 ul 10% APS

2 ul TEMED

Polymerize both the stacking and separating gels at the same time (I used small disposable tubes), and pour stacking gel directly onto the separating gel (pour carefully but quickly--they won't mix but the separating gel polymerizes within 2-3 minutes). Make each separating gel mixture separately and add TEMED and APS right before pouring. Multiple stacking gel mixtures can be made in the same tube, but you have around 10 minutes before these start to polymerize too.

Sample loading and electrophoresis:

For the minigels, 5-10 ul per well gives best results. Layer the samples in the well very carefully, and be sure to flush out any unpolymerized acrylamide before loading.

Electrophorese at 25-35 mA (constant current) per gel in minigel setup. Foam will appear on the top (cathode), and additional cathode buffer may have to be added during the run.

15% tricine-PAGE