Tricine-SDS-PAGE Protocol

Tricine-SDS Page Protocol

The following reagents are needed in advance:

Gel monomer (30% acrylamide, 29:1). 29.0 g acrylamide and 1.0 g bisacrylamide are dissolved in a total volume of 100 mL MQ H2O. Store at 4 °C, protect from light.

Gel Buffer. Dissolve 181.7 g Tris base and 1.5 g SDS in 400 mL MQ H2O. Adjust the pH to 8.45 using concentrated HCl. Bring to a final volume of 500 mL with MQ H2O. Store at 4 °C.

Anode Buffer (Lower Buffer). Dissolve 12.11 g Tris base in 400 mL MQ H2O. Adjust to pH 8.9 with HCl. Bring to 500 mL with MQ H2O. Store at 4 °C

Cathode Buffer (Upper Buffer). Dissolve 6.055 g Tris base and 8.96 g Tricine in a total volume of 500 mL MQ H2O. Add 0.5 g SDS. Store at 4 °C.

NOVEX 2X Tricine-SDS sample buffer. For 10mL, mix the following:

3.0 mL 3.0 M Tris-HCl, pH 8.45
2.4 mL glycerol
0.8 g SDS
1.5 mL 0.1% Coomassie Blue G
0.5 mL 0.1% Phenol Red
Distilled water to 10 mL
Other Reagents Needed: TEMED (pure liquid), 10 % (w/v) ammonium persulfate (made freshly), 0.1% (w/v) SDS, and glycerol.

Gel Preparation

Prepare & Pour the Separating Gel: Note - enough for four Novex 1.0 mm mini-gel cassettes. This recipe is for a 12.0 % running gel. In a disposable 50 mL conical tube, mix the following:

12.0 mL of gel monomer.
10 mL of gel buffer
3.1 mL glycerol
4.8 mL MQ H2O
0.1 mL 10% ammonium persulfate.
Iniatiate polymerization by adding 40 uL of TEMED. Transfer to the gel cassettes; bring to approximately 1.5 cm from the top of gel blank (which is reserved for the stacking gel) and gently overlay with a small volume (ca. 0.5 mL) of 0.1% SDS. Polymerize for 30 min. Remove layer of SDS after the gel has polymerized.

Prepare & Pour the Stacking Gel: Note - enough for four Novex 1.0 mm mini-gel cassettes. This recipe is for a 4.0 % stacking gel. In a disposable 50 mL conical tube, mix the following:

1.25 mL of gel monomer
2.5 mL of gel buffer
6.25 mL H2O
0.05 mL 10% ammonium persulfate
Iniatiate polymerization by adding 20 uL of TEMED. Transfer to the gel cassettes and insert comb. Clamp along the top with binder clips to insure well formed wells. Polymerize for 30 min.

Running, Developing and Drying the Gel.

Sample Preparation Samples are prepared by mixing a equivalent volume of sample with an equivalent volume of NOVEX 2X Tricine-SDS sample buffer. The samples are then heated for 5 min in a boiling water bath, centrifuged for one minute,

Gel Setup and Run Parameters. Prior to loading, fill the upper buffer chamber with cathode buffer and the lower chamber with 250 mL of anode buffer. Electrophorese at constant current - 15 mA until the samples have migrated through the stack; 30 mA for the remainder of the run (until the tracking dye has migrated out from the lower slot).

Staining. Staining is accomplished by soaking the gel in a solution of 45% methanol, 45% deionized water, 10% acetic acid, and 0.25% (w/v) Coomassie Blue R-250 on a table top shaker for 30 minutes. The gel is then destained by soaking in a solution of 45% methanol, 45% deionized water, and 10% acetic acid on a table top shaker. Periodically replace with fresh solution until desired destaining is achieved.

Gel Drying. After destaining, the gel is prepared for drying by soaking it for five minutes in gel drying solution (5% glycerol/30% methanol). Next, soak a single piece of cellophane in the same gel dry solution for 30 seconds. Lay down the cellophane first, and the gel on top of this. Next soak the second piece of cellophane in gel dry solution for 30 seconds, and then overlay this on the gel to make a sandwich. Squeeze out any air bubbles and then clamp on upper frame. Dry at room temperature.

Other Protocols Links:

Tricine–SDS-PAGE

Hermann Schägger¹

http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.4.html 

Tricine-SDS-PAGE

Protocol adapted from Schägger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368-379, as modified by Lesse, A. J., et al. 1990. Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Immunol. Methods 126:109-117.

http://wheat.pw.usda.gov/~lazo/methods/goldberg/tricine.html

Tricine-SDS-PAGE(ref Schagger, H., and von Jagow, G., Analytical Biochem. 1987, 166, 368-379)

http://molecular-neurogenetics.mcgill.ca/PDF/PDF%20Protocols/Tricine_SDS_PAGE.pdf.

 

17.5% Tricine SDS-PAGE Protocol(from Dr. Chen-ya Chien)

http://www-nmr.cabm.rutgers.edu/labdocuments/biochemprotocols/sds.html