Blotting Procedures

Western, Northern & Southern Blots

Blotting describes a process that transfers molecules separated by electrophoretic procedures on agarose or polyacrylamide gels to a special membrane that is better suited to support subsequent investigations.

DNA blots are called southern blots after the inventor Edward Southern. Because the basic principle behind DNA blots is very similar to those for protein and RNA blots, these procedures were named western and northern blot accordingly.

Western Blots

After separating proteins on a gel, they are transferred onto a membrane made of nitrocellulose or PVDF by applying pressure or electrical current. Part of the protein contained in the gel transfers to the membrane. The transfer or blotting process is driven by hydrophobic interaction between the membrane and the protein countered by similar interactions between the gel and the protein. Hence, the protein is not completely transferred to the membrane. The reason why this is important is shown below. The relative location present on the gel must be maintained in the blotting process.

After transferring of the protein, the membrane surface is deactivated or “blocked” by treatment with a bulk protein solution, usually consisting of a solution of milk powder. The membrane can now be subjected to immunostaining typically by applying an indirect ELISA protocol. One (or multiple) primary antibody is applied that is specific for the protein to be visualized on the membrane.

Following several washing steps, a secondary conjugated antibody is applied that recognizes the constant region of the primary antibody. The conjugate is typically a marker enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). Alternatively, other markers such as biotin or digitoxigenin (DIG), fluorescence markers or radiolabels can be employed. Marker enzymes do have the advantage of signal amplification, which provides additional sensitivity to the assay. The secondary antibody can be visualized by the application of the corresponding staining solution. Radiolabeled antibodies will show in a radiogram.

In parallel, the electrophoresis gel is stained with a protein staining reagent: this is why it is important that some protein remains in the gel. Comparison with the unspecific bands on the gel to the (hopefully) specific bands on the membrane allows for assignment. This method can be used in an inverse manner for the identification of antibodies rather than protein. One important application of this procedure is in HIV diagnostics.

Southern Blots

This method is used for the identification of DNA fragments that are separated on an 1% agarose electrophoresis gel. In principle it is similar to the western blot only that we are using labeled DNA to detect the denatured DNA transferred from the gel to the blotted membrane. The crucial step is to take an “image” of the gel before transferring the DNA in the gel to a nylon membrane. This transfer (or blotting) is supposed to maintain the relative location of all DNA fragments and this will only work after denaturing the DNA fragments with sodium hydroxide (breaking up double strands). This may require the application of pressure or other methods to proceed to completion. In addition, small concentrations on the gels should show on the blot.

The single stranded DNA fragments on the membrane are then immobilized by heat or UV light exposure. Detection of specific sequences occurs by hybridization. A sequence of DNA or RNA, which carries a detectable label, complementary to the DNA fragment of interest is applied to the membrane. Chromogenic, fluorogenic or radiolabels can be used.

After washing and the application of the corresponding staining reagent, hybridized bands on the membrane are visible and they can then be correlated with the bands on the original gel.

Northern Blots

The northern blot is similar to the southern blot except that it is used to detect RNA. Mapping RNA is especially important when studying gene expression. The process (except for the denaturation) is almost the same for RNA and DNA, except that the natural instability of RNA requires much more care.

As in the southern blot, the hybridization probe may be DNA or RNA carrying an appropriate detection label.

There are similar reverse blotting protocols for northern blotting, along the lines explained earlier for western blotting with regards to HIV: a library of known RNA fragments can be immobilized on a membrane; this membrane is then hybridized to a sample containing unknown RNA fragments.

Selection of Biosynth's Substrates for Alkaline Phosphatase:
Cat.No. - Product Name
B-7450 - 5-Bromo-4-chloro-3-indoxyl phosphate, disodium salt sesquihydrate
B-7452 - 5-Bromo-6-chloro-3-indoxyl phosphate, disodium salt monohydrate
B-7453 - 5-Bromo-6-chloro-3-indoxyl phosphate, disodium salt trihydrate
B-7500 5-Bromo-4-chloro-3-indoxyl phosphate, p-toluidine salt
B-7550 - 5-Bromo-6-chloro-3-indoxyl phosphate, p-toluidine salt
C-5100 - 6-Chloro-3-indoxyl phosphate, p-toluidine salt
I-6200 - 3-Indoxyl phosphate, bis(2-amino-2-methyl-1,3-propanediol) salt
I-6250 - 3-Indoxyl phosphate, disodium salt
I-6300 - 3-Indoxyl phosphate, p-toluidine salt
M-5800 - 4-Methylumbelliferyl phosphate, free acid
M-5850 - 4-Methylumbelliferyl phosphate, bis(2-amino-2-methyl-1,3-propanediol) salt
M-5900 - 4-Methylumbelliferyl phosphate, bis(cyclohexylammonium) salt
M-5920 - 4-Methylumbelliferyl phosphate, dilithium salt
M-5950 - 4-Methylumbelliferyl phosphate, disodium salt trihydrate

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