BLOTTING TECHNIQUE
Objective:- Demonstration of Blotting Technique.
General principle:-
> All the three blotting methods are fairly simple and usually consist of four separate steps:
1. Electrophoretic separation of protein or of nucleic acid fragments in the sample,
2. Transfer to and immobilization on paper support,
3. Binding of analytical probe to target molecule on paper, and
4. Visualization of bound probe.
> Molecules in a sample are first separated by electrophoresis and then transferred on to an easily handled support medium or membrane. This immobilizes the protein or DNA fragments, provides a faithful replica of the original separation, and facilitates subsequent biochemical analysis.
> After being transferred to the support medium the immobilized protein or nucleic acid fragment is localized by the use of probes, such as antibodies or DNA, that specifically bind to the molecule of interest. Finally, the position of the probe that is bound to the immobilized target molecule is visualized usually by autoradiography.
Procedure:-
1. Run the DNA/RNA/Protein gel to desired distance.
2. Visualize gel on UV box.
3. Soak gel in 0.2 N HCl until the bromophenol blue band begins to change color. (About 10 min usually). Alternatively, expose to UV transilluminator for 90 sec.
4. Rinse once with H2O.
5. Soak in 0.4N NaOH/0.6M NaCl for 30 min.
6. set up a transfer pyramid: put a glass plate on a support in a tray containing 1 litre of 0.5 M NaOH, and cover it with two sheets of filter paper (Whatman 3MM or other suitable paper) such that on all sides the filter paper contacts the solution and functions as a wick.
7. Wet the filter paper with 0.5 M NaOH and remove air bubbles by gently rolling a glass pipette back and forth on the paper.
8. Put the gel on the filter paper. Place a piece of nylon membrane slightly larger than the gel (prewet in 0.5 M NaOH) on the gel. Put 4pieces of prewet filter paper cut to size. Make sure to remove air bubbles between each layer.
9. Put a 10 cm stack of dry filter paper or paper towels, cut to size, on top of the pyramid. Place a glass plate and a weight of about 0.5 Kg on top.
10. The orientation of the gel and the nylon filter should be marked, for instance by cutting off a corner. Place strips of parafilm or plastic wrap around the gel to prevent direct contact between the stack of filter paper and the wick causing a short-circuit in the flow of the transfer solution. Allow the transfer to proceed for 12 h or longer.
11. Carefully disassembles the pyramid.
12. Remove membrane from blotter. Discard gel, whatman, and paper towels.
13. Rinse membrane in 2X SSC. Blot dry on a piece of whatman.
14. Sandwich blot between two pieces of dry whatman and bake in vacuum oven for 1 hour.
