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Methodology for Amino Acids and Proteins

 
     
 
Protein (Western) Blotting
 
The transfer of protein bands from an acrylamide gel onto a more stable and immobilizing support is called as protein blotting. A number of supporting matrices such as nitrocellulose, diazobenyloxymethyl cellulose sheets, nylon filters etc are used for the purpose. The separated proteins are buried in polyacrylamide gels and therefore further analysis of proteins or their recovery is cumbersome. However, the proteins can be efficiently transferred from the gel to the supporting medium by blotting. A variety of analysis involving immunoblotting, DNA-binding proteins, and glycoproteins could then be performed on the proteins blotted onto the filters. This method is an extension of the ‘Southern blot’ used to transfer DNA from gels to nitrocellulose filter and is called as the ‘Western blotting’. The beneficial of a protein blot include rapid staining/destaining detectio9n of proteins at low concentrations and rapid localization of the proteins in preparative gels. The blots can be preserved as replica of the original gels. The transfer of proteins is carried out either by electrophoresis (electroblotting) or by the capillary action of buffer (capillary blotting). The latter method is described below.
 
 


Principle
The separated proteins are transported out of the gel either by the capillary action of the buffer or in an electric field. The presence of SDS increases the solubility of proteins and thus facilitates the migration of proteins. Once out of the gel, the proteins comes in contact with the nitrocellulose membrane which binds the protein very strongly onto the surface as a thin band thus producing a replica of original gel.
 
 
Materials
Nitrocellulose Paper (pore size 0.20-0.45mm)
Blotting Buffer (pH 8.3)
0.02M Tris-HCl           2.42g
0.15MGlycine             10.25g
20% Methanol            200mL
Water to                       1L
(can be stored at 4°C for 2-3weeks).
Protein Stain: 0.01% Amido black 10B in Methanol:Acetic acid:Water (5:1:5)
Destaining Solution: Methanol:Acetic acid:Water (5:1:5)
 


 
Procedure
1.
Arrange a platform by placing a 25 x 20cm glass plate at a suitable height on the work bench.
2.
Assemble six layers of Whatman No. 1 filter paper and place over the platform. Dip the short ends of the papers in two glass trays containing the blotting buffer placed on either side of the platform. Let the papers wet thoroughly. The papers should be wide enough to accommodate the gel to be blotted.
3.
After the separation of proteins in slab gels by SDS-PAGE, discard the stacking gel and carefully lay the separation gel to be blotted on the wetted filter paper. Cut the required portion of the gel with a scalpel blade if the whole gel is not to be blotted, and lay carefully.
4.
Take a piece of nitrocellulose paper exactly the same size of the gel to be blotted. Wet thoroughly by floating on the blotting buffer. Carefully place this wetted nitrocellulose paper over the gel without trapping any air bubbles. This is conveniently done by lowering first the middle of the paper on the gel and then laying towards the ends. It is again preferable to wet the top of the gel with blotting buffer before layering the nitrocellulose paper.
5.
Cut out the middle of a cling film to the size of the gel, and lay it over the nitrocellulose paper such that the film does not cover the nitrocellulose paper. In other words, the cling film will form a frame revealing the nitrocellulose paper covering the surrounding portions. This shall prevent by-pass of buffer from the bottom layers to top layers of filter papers directly. This is to ensure on any account the buffer should pass only through the gel.
6.
On top of the film, lay six layers of Whatman 3mm filter paper cut to the same size as the gel followed by a way of absorbant material (tissue paper or disposable nappy) also of the same size of the gel.
7.
Place a heavy weight for example a conical flask containing one liter water, over the sandwich and leave the set up for 1-2 days. There should be ample blotting buffer in the tanks during blotting.
8.
The buffer from the bottom layer of filter papers moves upward by absorption via the gel and nitrocellulose paper. During this process, the proteins are transferred by capillary action from the gel to the nitrocellulose which has more attraction to the protein.
9.
After blotting for required period, recover the nitrocellulose paper disassembling the set-up. The nitrocellulose paper can be stored pressed between a fold of filter papers until required for further analysis or can be stained directly.
10.
Immerse the protein blot in the amido black dye solution for 10-15min with gentle shaking. Subsequently, destain the blot in the destaining solution with repeated changes. The transferred proteins are visualized as black bands. The blot is then dried between several sheets of filter paper held flat with a heavy weight.
 
 
Notes
1.
The nitrocellulose sheets should be stored air tight at 4°C to prevent them being contaminated by volatile chemicals etc.
2.
While assembling the sandwich for blotting care must be taken to see that there is no direct contact of nitrocellulose paper or the above layers with the bottom layers of filter paper in order to avoid the flow of the buffer directly. It should only flow through the gel to the nitrocellulose paper in an ideal blot assembly.
3.
Nitrocellulose paper should be thoroughly wetted before being used.  Any bubbles between the gel and nitrocellulose paper will result in poor transfer of proteins.
4.
Capillary blotting is a passive process and requires longer period (2-4 days). The duration of blotting largely depends upon various factors such as the porosity of nitrocellulose, percentage of acrylamide and thickness of the gel, the ionic strength of the blotting buffer, the solubility of proteins etc. the low MW proteins are easily transferred than the high MW proteins.
5.
The transfer of proteins by electroblotting is much faster and efficient than the capillary blotting method. Suitable cassettes to assemble the sandwich for electro-blotting are commercially available.
6.
Equilibrium of the gel prior to blotting for renaturing of the separated proteins is suggested depending upon the further analyses of the blot. The gel is equilibrated in the following buffer with constant shaking for 30-60min prior to capillary blotting for renaturing the separated proteins.
1M Tris-HCl pH 7.0              5mL
5M NaCl                              5mL
0.1M EDTANa2                   10mL
0.1M Dithiothreitol              0.5mL
Urea                                   120.12g
Water to                             350mL
7.
Wear gloves while handling the gel and nitrocellulose paper to avoid contamination. Laying a second nitrocellulose paper at the bottom of the gel is also a common practice in contact-diffusion blotting. By this way two blots are obtained simultaneously.
 
 
References
1. Towbin, H, Staehelin, T and Gordon, J (1979) Proc Natl Acad Sci USA 76 4350.
2. Burnetter, W N (1981) Anal Biochem 112 195.
3. Manual on ‘Techniques in Molecular Biology’ (Proteins) (1986) Workshop held at the Department of Biochemistry Tamil Nadu Agricultural University Coimbatore p 51.
 
     
 
 
     




     
 
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