TEMED 3 5

Sum 5000 5000

(AP is Ammonium persulfate)

Or:

Reagents Amount (ml)

Separating gel (%12) Stacking gel (4%)

Water 3344 3020

Tris-HCl 2500 (1.5 M, pH 8.8) 1250 (0.5 M, pH 6.8)

SDS (10%) 100 50

Acr-Bis (30%) 4000 650

AP 50 25

TEMED 6 5

Sum 10000 5000

(AP is Ammonium persulfate)

3. Take 50 ml of sample and add an equal volume of 2 x SDS gel-loading buffer.

4. 2 x SDS gel-loading buffer:

100 mM Tris-HCl (pH 6.8)

200 mM dithiothreitol

4% SDS

0.2% bromophenol blue

0.2% glycerol

2 x SDS gel-loading buffer lacking dithiothreitol can be stored at room temperature. Dithiothreitol should then be added, just before the buffer is used, from a 1 M stock (Dissolve 3.09 g of dithiothreitol

in 20 ml of 0.01 M sodium acetate (pH 5.2). Sterilize by filtration. Dispense into 1-ml aliquots and store at –20^oC).

5. Boil the sample (in loading buffer) at 100^oC for 3 – 5 minutes.

6. Load the sample for electrophoresis: 8 V/cm (6 x 8 = 48 volts) before the bromophenol blue (dye) front has moved into the resolving gel and 15 V/cm (6 x 15 = 90 volts) until the bromophenol blue reaches the bottom of the resolving gel.

7. Make the gel for transfer in transfer buffer: 0.65 mA/cm2 (about 100 volts) for 1.5 – 2 hours, or 30 volts overnight, on ice.

8. Western blotting transfer buffer:

Western Transfer Buffer, 1000 ml
Tris	48 mM	5.814 g
Glycine	39 mM	2.928 g
SDS	0.037%	0.37 g
Methanol	20%	200 ml

SDS: 3.7 ml of 10% SDS

9. Block the filter with blocking buffer for 1 – 2 hours at room temperature (0.1 ml blocking solution per cm² filter), with gentle agitation on a platform shaker.

10. Blocking solution:

Blocking solution, 100 ml, in PBS
Nonfat dried milk	5%
Antifoam A	0.01%
Sodium azide	0.02%

Sodium azide: 1 ml of 2% solution

11. Phosphate-buffered saline (PBS), pH 7.4, 1000 ml:

NaCl	8 g
KCl	0.2 g
Na₂HPO₄	1.44 g
KH₂PO₄	0.24 g
Adjust to pH 7.4 with HCl

12. Discard blocking solution and immediately incubate filter with primary antibody:

A. Add 10 ml (0.1 ml of blocking solution per cm² of filter).

B. Blocking solution:

Blocking solution, 10 ml, in PBS (pH 7.4)
Nonfat dried milk		5%
Antifoam A		0.01%
Sodium azide		0.02%

C. Add 0.005 ml of primary antibody (1:2000) in to blocking solution.

D. Incubate at 4^oC for 2 hours or overnight with gentle agitation on a platform shaker.

13. Discard blocking solution and wash filter 3 times (10 minutes each time) with 250 ml of PBS.

14. Incubate the filter with 150 mM NaCl, 50 mM Tris-HCl (pH 7.5) (phosphate-free, azide-free blocking solution) for 3 times for 10 minutes each time.

15. Immediately incubate the filter with secondary antibody:

A. Add 10 ml of phosphate-free, azide-free solution (150 mM NaCl, 50 mM Tris-HCl, 5% nonfat dry milk pH 7.5).

B. Phosphate-free, azide-free blocking solution

(pH 7.5, 1000 ml):

NaCl	150 mM	8.766 g
Tris-HCl (pH 7.5)	50 mM	6.057 g
12 N HCl		about 3.35 ml
Nonfat dried milk	5% (w/v)

C. Add 0.005 ml of secondary antibody solution (1:2000).

D. Incubate 1 – 2 hours at room temperature with gentle agitation.

E. Discard secondary and wash with 150 mM NaCl, 50 mM Tris-HCl (pH 7.5) (phosphate-free, azide-free solution) for 3 times for 10 minutes each time.

16. Alkaline phosphatase satin:

A. Add 5 ml of the substrate 5-brono-4chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) solution (Sigma).

B. Observe the filter for the blue color on the filter (about 20 minutes).

C. Discard BCIP/NBT solution when the bands are clear (about 20 minutes).

D. Immediately stop the enzymatic reaction by add water.

E. Cover the filter with plastic membrane and keep the filter.

F. Analyze the blue bands and compare the color.

The half-life of a 0.02 mM aqueous solution of PMSF is about 35 minutes, at 8.0 pH. PMSF is usually stored as a 10 mM or 100 mM stock solution (1.74 or 17.4 mg/ml in isopropanol) at –20^oC.

1X SDS gel-loading buffer lacking dithiothreitol can be stored at room temperature. Dithiothreitol should then be added, just before the buffer is used, from a 1 M stock (Dissolve 3.09 g of dithiothreitol in 20 ml of 0.01 M sodium acetate (pH 5.2). Sterilize by filtration. Dispense into 1-ml aliquots and store at –20^oC).

III. Western Blotting Solutions:

Tissue Extract buffer, 100 ml

50 mM Tris-HCl (pH 8.0), 0.6 g (Or 50 mM HEPES (pH 7.0), 1.19 g)

150 mM NaCl, 0.88 g

0.02% sodium azide, 0.02 g

0.1% SDS, 0.1 g

0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF), 0.01 g

0.001 mg/ml aprotinin, 0.1 mg

1% Nonidet P-40 (NP-40), 1 ml (Or 1% Triton X-100, 1 ml)

2 X SDS gel-loading buffer, 100 ml

100 mM Tris-HCl (pH 6.8), 1.21 g

200 mM dithiothreitol

4% SDS, 0.4 g

0.2% bromophenol blue, 0.2 g

20% glycerol, 20 ml

1.5 M Tris-HCl, pH 8.8, 300 ml

Tris, 54.5 g

HCl, 12 N, 6.375 ml

0.5 M Tris-HCl, pH 6.8, 300 ml

Tris, 18.17 g

HCl, 12 N, 11.5 ml

SDS 5x Running Buffer, pH 8.3, 1000 ml

Tris, 125 mM, 15.14 g

Glycine, 1.25 M, 93.84 g

SDS, 0.50%, 5 g

Western Transfer Buffer, 1000 ml

Tris, 48 mM, 5.814 g

Glycine, 39 mM, 2.928 g

SDS, 0.04%, 0.37 g, 3.7 ml of 10% SDS

Methanol, 20%, 200 ml

Blocking solution, 100 ml, in 100 ml phosphate-buffered saline (PBS, pH 7.4)

Nonfat dried milk, 5%, 5 g

Antifoam A, 0.01%, 10 ml

Sodium azide, 0.02%, 20 mg

Phosphate-buffered saline (PBS), pH 7.4, 1000 ml (adjust to pH 7.4 with HCl)

NaCl, 8 g

KCl, 0.2 g

Na₂HPO₄, 1.44 g

KH₂PO₄, 0.24 g

Phosphate-free, azide-free blocking solution, 1000 ml (adjust pH with 12 N HCl about 3.35 ml)

150 mM NaCl, 8.766 g

50 mM Tris-HCl (pH 7.5), 6.057 g

5% (w/v) nonfat dried milk