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Como se da el Western Blot Protocol


Enviado por   •  18 de Febrero de 2018  •  Apuntes  •  1.044 Palabras (5 Páginas)  •  124 Visitas

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WESTERN BLOT PROTOCOL

A) Preparing the gel

Choose the correct gel percentage depending on the protein size.

Protein size (kDa)

Gel percentage (%)

4 – 40

20

12 – 45

15

10 – 70

12.5

15 – 100

10

25 – 200

8

Recipes for the different gel percentages

1. – Separating gel (20 ml)

Reactives

8%

10%

12.5%

15%

20%

ddH2O

9.2 ml

7.9 ml

6.2 ml

4.6 ml

1.2 ml

Acrylamide 30%

5.33 ml

6.67 ml

8.33 ml

10 ml

13.33 ml

Tris 1.5 M pH 8.8

5 ml

5 ml

5 ml

5 ml

5 ml

10% SDS

200 μl

200 μl

200 μl

200 μl

200 μl

10% APS

200 μl

200 μl

200 μl

200 μl

200 μl

TEMED

20 μl

20 μl

20 μl

20 μl

20 μl

2. – Stacking gel (100 ml)

Reactives

4%

6%

ddH2O

60 ml

52 ml

Acrylamide 30%

13.4 ml

20 ml

Tris 0.5 M pH 6.8

25 ml

25 ml

10% SDS

1 ml

1 ml

10% APS (3.5 ml)

35 μl

35 μl

TEMED (3.5 ml)

3.5 μl

3.5 μl

B) Protein separation by gel electrophoresis

1. – Load equal amounts of protein (20 μg) into the wells of the gel.

2. – Run the gel for 5 minutes at 50 V.

3. – Increase the voltage to 100-150 V to finish (The higher the separating gel percentage, the more time the gel will take to finish).

C) Transferring the protein to the membrane

  • For large proteins (larger than 100 kDa) it is recommendable to use the wet transfer and to eliminate methanol from the transfer buffer. Methanol in the transfer buffer improves binding proteins to nitrocellulose only. Elimination of methanol results in increased transfer efficiency but diminishes binding to nitrocellulose. Transfer efficiency is increased because methanol causes gel pores to contract resulting in capture of large molecular weight proteins within the gel matrix. Use of PVDF membrane for protein transfers eliminates the methanol requirement, and constitutes a logical strategy for analysis of high molecular weight or difficult-to-transfer proteins. Also it is recommended to include SDS to the transfer buffer at a final concentration of 0.1%.
  • For small protein (smaller than 100 kDa) it is recommendable to use the transfer buffer with 20% of methanol and without SDS.
  • Nitrocellulose membrane: No pre-activation is required. Low molecular weight proteins (>15 kDa) may be lost during post transfer washes, thus limiting detection sensitivity. Smaller pore size nitrocellulose membrane (0.2 μm), has been shown to be effective in eliminating this loss. Larger proteins (> 100 kDa) denatured by SDS may transfer poorly due to the addition of methanol to the transfer buffer. Methanol increases binding of SDS-proteins to nitrocellulose, but decreases pore sizes in the gel. Elimination of methanol from SDS-protein transfers results in considerably diminished binding. Adding SDS (up to 0.1%) to the transfer buffer increases the transfer efficiency of proteins, but reduces the amount of binding to the membrane.
  • PVDF membrane: Polyvinylidene diflouride (PVDF) retains proteins under extreme conditions of exposure of exposure to acidic or basic conditions, and in the presence of organic solvents. PVDF membrane exhibits better binding efficiency of blotted material in the presence of SDS in the transfer buffer. The membrane must first wetted in 100% methanol but can then be used in buffer, which does not contain it.

1. – Place the gel in transfer buffer for 15-20 min (equilibrating).

Wet Transfer

2. – Cut the membrane and the filter paper to the dimensions of the gel. Always wear gloves to prevent contamination.

3. – Prepare the gel sandwich:

  • Place the cassette, with gray side down, on a clean surface.
  • Place one prewetted fiber pad on the gray side of the cassette.
  • Place a sheet of filter paper on the fiber pad.
  • Place the equilibrated gel on the filter paper.
  • Place the prewetted membrane on the gel.
  • Remove any air bubbles which may have formed for good results.
  • Complete the sandwich by placing a piece of the filter paper on the membrane.
  • Add the last fiber pad.

4. – Close the cassette firmly, being careful not to move the gel and filter paper sandwich. Lock the cassette closed with the white latch.

5. – Place the cassette in module. Place it tank and fill to the blotting mark on the tank.

6. – Add a standard stir bar to help maintain even buffer temperature and ion distribution in the tank.

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