Upload Now
In this article we will discuss about the principle, requirements and procedure for capillary blotting.
Principle:
Samples of proteins isolated on gel are transported out of gel by capillary action of buffer. The solubility of proteins increases by the presence of SDS and helps its migration to nitrocellulose membrane which binds it strongly on its surface which in turn facilitates further analysis.
Requirements:
1. Nitrocellulose paper (pore size 0.20- 0.45 pm) at 4°C
2. Blotting buffer (pH 8.3):
Tris HCl 0.02M: 2.42 g
Glycine 0.15M : 10.25 g
Methanol 20% : 200.00 ml
Distilled water: 1000.00 ml
Store at 4°C: omit methanol
for nylon filters
3. Protein stain: 0.01%
Amido black: 10B in 100 ml of
Methanol: acetic acid : water 5:1:5
4. Destaning solution
Methanol : acetic acid : water 5:1:5
5. Equilibration buffer:
Tris HCl (pH 7.0) 1 M: 5.0 ml
NaCl 5 M: 5.0 ml
EDTA Na2 0.1 M 0.0 ml
Dithiothreitol 0.1 M: 0.5 ml
Urea: 120.2 g
D. Water upto: 350.0 ml
6. Whatman No 1 filter papers.
7. Whatman 3 mm filter papers.
8. Gel with protein bands.
9. Glass plates.
10. A weight of approximately 500 g.
11. Tray for buffer.
Procedure:
1. Equilibrate the gel with protein in equilibration buffer, prior to capillary blotting, for 30-40 minutes with constant shaking.
2. Place a 25 X 20 cm glass plate to make a platform at a suitable height and place six layers of Whatman No. 1 filter paper on it.
3. Place two glass trays with blotting buffer on either side of the platform. Dip the short ends of the paper in buffer and allow the papers to wet completely.
4. Keep the equilibrated gel with protein on the wetted filter paper carefully.
5. Wet a piece of nitrocellulose paper exactly of the same size of the gel that is to be blotted and wet it thoroughly by floating it on the blotting buffer.
6. Carefully place this wetted nitrocellulose paper on top of the gel, taking care to see that no air bubbles are trapped in between. For this, lower first the middle portion of the paper on the gel and then gently lower the sides and end. Before placing the paper, wet the surface of the gel with blotting buffer.
7. The edges of the nitrocellulose paper, if it clings down, should be cut to prevent by-pass of buffer from bottom directly to the top layers of filter papers in order to ensure that buffer should pass only through the gel.
8. Lay six layers of Whatman 3 mm filter paper, cut to the same size as that of the gel, on the paper and a stack of absorbant tissue paper on top, all of the same size as that of the gel.
9. Place a weight of approximately 500 g over the set up and leave for 1—2 days. Plenty of blotting buffer should be there in the trays.
The buffer moves from the bottom layers of the filter paper upwards via the gel to the nitrocellulose paper. During this movement proteins are transferred from the gel to the nitrocellulose paper by capillary action.
10. After the required period, recover the nitrocellulose paper, press it in between filter papers and store until required or can be stained.
Staining:
Staining can be done by immersing the protein blot in amino black dye solution for 10-15 minutes with gentle shaking. Destain with repeated changes, in destaining solution and observe black bands of protein. Dry the blot between folds of filter paper and place it under heavy weight.
Electro-blotting:
The entire set of sandwitch can be electro-blotting with gel-side facing the cathode and nitrocellulose paper side facing the anode. Blotting is carried out overnight at 20 mA in a cold room.