Buffer recipe

Protocols

Western Blot

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WESTERN BLOT:

Solutions:

a) 1.5 M Tris-HCl pH 8.8 Dissolve 54.5 g Tris base in 200 ml of distilled water. Adjust pH to 8.8 using concentrated HCl. Make up total volume to 300 ml with distilled water. Store at 2-8 C.

b) 0.5 M Tris-HCl pH 6.8 Dissolve 12.1 g of Tris base in 150 ml of distilled water. Adjust pH to 6.8 using concentrated HCl Make up total volume to 200 ml with distilled water. Store at 2-8 C

c) 10% (w/v) Sodium dodecyl sulfate (SDS) Dissolve 5 g of SDS in 50 ml of distilled water. Mix until dissolved. Store at room temperature.

d) 10% Bromphenol blue Dissolve .1 g in 1 ml of distilled water. Store at room temperature.

e) SDS-PAGE loading buffer In a 20 ml vial mix the following: 4.2 ml water 1.0 ml 0.5 M Tris-HCl pH 6.8 0.8 ml glycerol 1.6 ml 10 % (w/v) SDS 0.4 ml 2-mercaptoethanol 0.02 ml 10% (w/v) bromphenol blue Store at room temperature

It is recommended that the above recipe is used because alternative tracking dyes or even an excess of dye will give fluorescent bands at the gel front, which may interfere with detection of the protein of interest.

f) 10% (w/v) Ammonium persulphate (AMPS) Dissolve 0.1 g of AMPS in 1 ml of water. USE IMMEDIATELY.

g) SDS-PAGE running buffer Dissolve 15 g of Tris base (25 mM), 72 g glycine (192 mM) and 5 g SDS (0.1% w/v) in 5 liters of distilled water. Mix until dissolved Store at room temperature.

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h) Transfer buffer Dissolve 15 g of Tris base (25 mM) and 72 g glycine (192 mM) in 4 liters of distilled water. Add 1000 ml methanol (20 % v/v) and mix thoroughly. Store at 2-8 C.

i) Tris buffered saline (TBS ) pH 7.6 20 ml 1 M Tris HCl pH 7.6 (20 mM) 8 g Sodium chloride (137 mM) Dilute to 1000 ml with distilled water- check pH. Store at room temperature.

j) TBS-Tween (TBS-T) Use for wash buffers and diluents. A 0.1% (v/v) Tween 20 concentration in TBS is suitable for most fluorescent Western blotting work on PVDF membrane but concentration ranging from 0.05% to 1 % may be required to suit your specific needs. Wash buffers should be stored at room temperature.

Flow diagram of procedure Separate protein sample by electrophoresis V Transfer to membrane V Block non-specific sites V Incubate in primary antibody V Incubate in alkaline phosphate linked anti-species antibody V Incubate in Vistra ECF substrate V Scan

Gel preparation

For 20 ml

Component 8% 10% 15%Water 9.3 ml 7.9 ml 4.6 ml 30% Acrylamide 5.3 ml 6.7 ml 10 ml mix (19:1) 1.5 M Tris HCl 5.0 ml 5.0 ml 5.0 ml pH 8.8 10 % (w/v) SDS 0.2 ml 0.2 ml 0.2 ml 10 % (w/v) AMPS 0.2 ml 0.2 ml 0.2 ml TEMED 0.012 ml 0.008 ml 0.008 ml

Stacking gel:

Component Water 5.6 ml 30 % Acrylamide mix (19:1) 1.7 ml 0.5 M Tris HCl pH 6.8 2.5 ml 10 % (w/v) SDS 0.1 ml 10 % (w/v) AMPS 0.1 ml TEMED 0.01 ml

Mix samples with loading buffer and boil for 5-10 min. After load on the gel and let separate with constant 20 mA. (If two gels 40 mA)

Performing the blotting:

After the electrophoresis build a sandwich out of gel and membrane: The PVDF membrane should be pre-wetted in methanol for 5 seconds and rinsed in water for 5 min to remove the methanol, then equilibrated in transfer buffer for 10 to 15 min.

Cut out some whatman paper in the same size like gel and membrane. Sandwich order: a) two or three whatman paper b) PVDF membrane c) Gel d) Two or three whatman paper

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The sandwich should be put in the blotting machine so that the membrane is on the side of the Cathode.

Blocking the membrane Remove the membrane from the apparatus. Block the nonspecific binding sites by immersing the membrane in 5% blocking reagent in Tris buffered saline Tween 20 (TBS-T) for one hour on an orbital shaker at room temperature

Optimum Tween concentration will vary to suit specific experiments, but a 0.1% Tween 20 concentration is suitable for most fluorescent work on PVDF membrane. Certain experimental situation may require alteration of the time and temperature of the blocking incubation. Alternatively, membranes may be left in the blocking solution overnight at 2-8 C.

Washing Wash membrane with TBS-T. Briefly rinse membrane twice with fresh changes of washing buffer. Then wash three times: 20 min, 10 min, 10 min. with fresh changes of washing buffer on an orbital shaker at room temperature The volume of wash buffer should be as large as possible. 4 ml of buffer per cm2 of membrane is suggested.

Primary antibody During the washing step dilute the primary antibody in TBS-T. (for 5-alpha reductase: 1:1000 ) Incubate the membrane in the diluted antibody for 1 hour on an orbital shaker at room temperature.

The required dilution of the primary antibody to give the optimum results will vary and should be determined for each antibody used. When quantifying protein samples linearity can, in some cases, be improved by decreasing the primary antibody concentration. Too high a concentration may lead to saturation of binding sites which will hinder the binding of the secondary antibody and should therefore be avoided. For impure antibodies the addition of blocking reagent to the primary antibody incubation may improve signal to noise ratio.

Washing s.a.

Secondary antibody Dilute the anti-rabbit-AP 1:10000 in TBS-T.

Incubate the membrane in diluted antibody for 1 hour on an orbital shaker at room temperature.

Washing s.a.

Substrate Use 24 ul substrate per cm2 membrane. Place a piece of saran wrap on the bench and using tissue to smooth it out ensuring there are no air bubbles or creases in it. Pipette the substrate on the saran wrap and lay the membrane with the protein side on the substrate (ensure that there are no air bubbles) Leave on the substrate for 5 min at room temperature.Do not move the blot during the incubation.

Scan Remove the membrane carefully from the substrate and place directly, protein side down on the scanner.

PVDF membranes can be stored at 2-8 C.

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