PROTOCOL

Casting the gel

Overlay the polymerized separating gel (10% or 16%, or 16%/6 M urea) directly with a 4% sample (stacking) gel prepared as indicated in the table in Step 1, except if resolution of proteins <5 kDa is desired. If resolution of proteins <5 kDa is desired, then use AB-6 instead of AB-3 for the separating gel and overlay the separating gel with a 1-cm 10% gel, made up as described in the table. The 16% separating gel and the overlaid 10% ‘spacer gel’ can be polymerized together if no glycerol is added to the 10% acrylamide gel mixture (the common role of glycerol in SDS gels is to increase the density of solutions and to facilitate gel casting; it has no obvious effect on protein separation). Introducing a 10% ‘spacer gel’ between 4% stacking and 16% separating gels considerably sharpens the bands for proteins and peptides of 1–5 kDa.

Sample preparation and protein loading

1.         For low-density samples such as elution fractions from chromatographic columns, add 5 µl of reducing or nonreducing sample incubation buffer A to 15 µl of sample.

2.         For high-density samples such as fractions from sucrose density gradients, add 5 µl of sample buffer B to 15 µl of sample.

3.         For pellet samples, resuspend the pellet in 15–20 µl of buffer A/4

※   Incubate samples at 37 °C for 15 min or for up to 60 min for samples that were in pellet form.

※   Avoid boiling samples, because membrane proteins can irreversibly aggregate in SDS at temperatures >50 °C.

Electrophoresis conditions

1.         Start electrophoresis with an initial voltage of 30 V and maintain at this voltage until the  sample has completely entered the stacking gel.

2.         The next appropriate voltage step can then be applied. The initial current may be as high as 80 mA for a 0.7-mm 10% gel.

※   Gels may warm up, but the temperature should not exceed 35–40 °C.

3.         Approaching the end of the run, voltage can be gradually increased to shorten the total time of electrophoresis.

※   Fast runs give better results than overnight runs, especially with 10% acrylamide gels.

※   Alternatively, a constant power of ∼10 W per gel might be set to ensure an even distribution of heat.