Resolving gel solution for 10% SDS-PAGE (18.5 cm x 20 cm gels)

 For unknown proteins, a 10 gel is an excellent choice for initial characterization.

 Use BIO-RAD, IPG Conversion Kit. This Kit allows 18.5 cm wide 20 cm long gels to be run on BIO-RAD’s PROTEAN II xi Cell thus accomodating commercially available immobilized pH gradient gel strips for 2-D second dimension protein separation.

 Clean the glass with ethanol and water. Assemble the set: insert a longer glass plate into a slot of clamps; place the spacers and insert a shorter inner glass plate; adjust the plates and spacers (the alignment card could be used for this purpose) so, that spacers and plates are flush at the bottom; tighten clamp screws and place ‘sandwich’ into a casting stand.

 Prepare: the flask with rubber stopper and box with ice. Keep a flask on ice during the preparation of solution.

 For the gels of 1 mm thickness:

 

80 ml (2 gels)

40 ml (1 gel)

Acrylamide/Bis-acrylamide (stock solution 30, w/v; crosslinker ratio 29:1; BIO-RAD, Cat.No: 161-0156)

26.64 ml

13.32 ml

TRIS-HCl 3M, pH 8.8

(0.372 M final)

9.92 ml

4.96 ml

Glycerol 50

(5 final)

8 ml

4 ml

Distilled H2O

34.64 ml

17.32 ml

SDS 10 (0.1 final)

800 microl

400 microl

 

Degas under vacuum for 15 min (do not forget to keep the flask with solution on ice). Then add:

TEMED (N,N,N,N’-Tetra-methylethylenediamine; BIO-RAD, Cat.No. 161-0800)

80 microl

40 microl

Ammonium persulfate 10, w/v

(0.01 final)

106.4 microl

53.2 microl

 

Fill the gel cassette with resolving gel solution and put some of iso-butanol on the top of the gel. After polymerization (at least after 30 min) aspirate an iso-butanol from the gel (use a syringe) or wash it with distilled water and fill the gel cassette with stacking gel solution.

 

Solutions: Tris T-1378 ‘Sigma’ 36.33g/100ml H2O, pH 8.8 with HCl

 

2002 Julius