Please Note: Do not substitute Tris-HCl for Tris Base or vice versa in the

     buffers used in Western Blotting.   If Tris-HCl is used instead

     of Tris base,   the concentration of salt will be too high and

     polypeptides will migrate anomalously through the gel,

     yielding extremely diffuse bands.

0.5 M Tris-HCl

Tris Base       3.03 g

- Dissolve in 30 mL ddH₂O.    pH to 6.8.    Q.S. to 50 mL mark with  ddH₂O.

- Autoclave.  Store at 4^o C.  Buffer will last 1 month.  After that bring to R.T.

   and check pH before using.

1.5 M Tris-HCl

Tris Base       9.09 g

- Dissolve in 30 mL ddH₂O.      pH to 8.8.     Q.S. to 50 mL mark with ddH₂O.

- Autoclave.  Store at 4^o C.  Buffer will last 1 month.  After that bring to R.T.

   and check pH before using.

2.5 M Tris-HCl

Tris-HCl          29.55g

- Dissolve in 40 mL  ddH₂O.      pH to 7.2.     Q.S. to 75 mL mark with  ddH₂O.

- Autoclave.  Store at 4^o C.  Buffer will last 1 month.  After that bring to R.T.

   and check pH before using.

N.B.  Do not substitute Tris Base for the Tris-HCl.   If Tris Base is used,  a large

         volume of concentrated acid  will be required to bring the pH down to 7.2.

Separating Gel      (7.5 % gel, 0.375 M Tris, pH 8.8)

ddH₂O                                                4.85 mL

1.5 M Tris-HCl <pH 8.8                    2.5 mL

10 % SDS                                          100 uL

Acrylamide/Bis (30%)                      2.5 mL

10% Ammonium persulfate50 uL

TEMED                                              5 uL

Total Volume                                   10 mL  (Enough for 2 mini-gels)

 •          Mix the first 4 ingredients in the order given.  Swirl gently to mix.

 •          To initiate polymerization,  add the ammonium persulfate and the TEMED;

            swirl gently to mix.  Cast the gel  immediately. 

N.B.    This gel is suitable for SDS-treated proteins in the approximate molecular

weight range of 40 - 250 kDa.  For proteins in the range of 10 -100 kDa,

use a 12% gel (See p.19 in the manual for recipe).

Stacking Gel    (4% gel, 0.125 M Tris,  pH 6.8)

ddH₂O                                                6.1 mL

0.5 M Tris-HCL, pH 6.8                    2.5 mL

10 % SDS                                          100 uL

Acrylamide/Bis (30%)                      1.33 mL

10 % ammonium persulfate50 uL

TEMED                                              10 uL

Total Volume                                   10 mL

 •          Mix the first 4 ingredients in the order given.  Swirl gently to mix.

 •          To initiate polymerization, add the ammonium persulfate and the TEMED;

            swirl gently to mix.    Cast the gel  immediately.

Acrylamide   (30% T, 2.67% C)

Acrylamide                                         14.6 g

N’N’-bis-methylene-acrylamide         0.4 g

- Dissolve in 30 mL ddH₂O.   Make up to 50 mL with ddH₂O.

- Filter.  Cover bottle with aluminum foil and store at 4^o C for a maximum

   of 30 days.

10 %  SDS   (Sodium Dodecyl Sulphate: aka Sodium Lauryl Sulphate)

10 g  SDS 

- Dissolve in  70 mL ddH₂O with gentle stirring; may need to heat to 68^o C to aid

  dissolution (Let cool to R.T before adjusting pH).

- pH  to 7.2

- Make up to 100 mL with ddH₂O.

N.B.    Wear a mask when weighing SDS and wipe down the area and balance

after use because the fine crystals of SDS disperse easily.

10% Ammonium Persulfate

- Dissolve 100 mg of ammonium persulfate in 1 mL ddH₂O.

- Prepare fresh daily.

Sample Buffer for Westerns  (Gels)

                        (Final Conc.)  4 mL               2 mL               1 mL

ddH₂O                                                                        1.9 mL            0.95 mL          475 uL

0.5 M Tris-HCl                       (62.5 mM)                  0.5 mL            250 uL            125 uL

Glycerol                                  (10%)                          400 uL            200 uL            100 uL

10% SDS                               (2%)                            800 uL            400 uL            200 uL

2-mercaptoethanol    (5%)                            200 uL            100 uL            50 uL

1% Bromophenol Blue         (0.05%)                      200 uL            100 uL            50 uL

•                      Mix 1 part  sample to  3  parts Sample Buffer.

•                      Boil samples for  5 min. and then quench on ice.

•                      Store Sample Buffer at R.T.

Sample Buffer for Western Dot Blots

                        (Final Conc.)  4 mL               2 mL               1 mL

ddH₂O                                                                        2.1 mL            1.05 mL          525 uL

0.5 M Tris-HCl                       (62.5 mM)                  0.5 mL            250 uL            125 uL

Glycerol                                  (10%)                          400 uL            200 uL            100 uL

10% SDS                               (2%)                            800 uL            400 uL            200 uL

2-mercaptoethanol    (5%)                            200 uL            100 uL            50 uL

•                      Mix  1 part  sample and 3  parts Sample Buffer.

•                      Boil samples for  5 min. and then quench on ice.

•                     Store Sample Buffer at R.T.

5x Running Buffer

Tris Base                   9.0 g

Glycine                       43.2 g

10% SDS                   30 mL

- Dissolve in 500 mL  ddH₂O.   Make up to 600 mL mark with ddH₂O.

- Check pH; it should be approximately 8.3.  However, do not adjust pH; if it

   is below 8.0,  remake buffer.

- Store at 4^o C.  Warm to R.T. before use if precipitation occurs.

Transfer Buffer

Tris Base (25 mM)                4.55 g

Glycine    (192 mM)   21.6 g

- Dissolve in 1.3 L ddH₂O.  Check pH; it should be approximately 8.3.  However,

   do not add acid or base to adjust it.  If  the pH is below 8.0, remake buffer.

- Make up to 1.5 L mark with ddH₂O.   Store at 4^o C.

Blocking Solution  (0.05 M Tris-HCl; 0.3% Tween 20; 5% Milk Powder)

Tris Base       3.03 gor        Tris-HCl          3.94 g

- Dissolve Tris in 400 mL  ddH₂O.      pH to 7.5.     Q.S. to 500 mL mark

   with  ddH₂O.

- Autoclave.    Add 1.5 mL Tween 20.  Store at 4^o C.                                  

- Just prior to using,   add milk powder:

To Make                    Add

20 mL            1.0 g

30 mL             1.5 g

40 mL             2.0 g

50 mL             2.5 g

Coomassie Blue Stain

ddH₂O                                    250 mL

Methanol                                200 mL

Glacial Acetic Acid                50 mL

Coomassie Blue Dye           0.5 g

- Mix  ddH₂O and methanol.  Add the acetic acid and then the dye.  Mix until

   the dye has dissolved.  Filter.  Store at R.T.

N.B.  Prepare in  fumehood.

Destain

ddH₂O                                    250 mL

Methanol                                200 mL

Glacial Acetic Acid                50 mL

-  Mix  ddH₂O and methanol.  Add the glacial acetic acid; mix.

-  Store at R.T.

N.B.  Prepare in  fumehood.