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The principle of silver nitrate staining of proteins is based on the reduction of silver ions to its metallic form. (Silver ions in alkaline conditions bind with proteins through å-amino group of lysine and sulphur groups of cysteine and methionine residues). When the complexed silver ions are reduced in the presence of formaldehyde, they become metallic silver, which is seen on the gel as bands. Staining of proteins with silver nitrate after SDS-PAGE is quicker and ~ 10 times more sensitive than Coomassie brilliant blue staining and that is the reason it is widely used in research laboratories these days.

The procedure is as follows:

Principle

Determination of the precise sequence of nucleotides in a sample of DNA is called “DNA Sequencing”. The most popular method for doing this is called the “Dideoxy method or Sanger Method” (named after Frederick Sanger, who was awarded the 1980 Nobel Prize in chemistry for inventing it). DNA is synthesized from four deoxynucleotide triphosphates. The top most figure is a normal nucleotide: deoxythymidine triphosphate (dTTP). During polymerization each new nucleotide is added to the 3' -OH group of the last nucleotide added.

The dideoxy method has been coined after the synthetic nucleotides that lack the –OH at the 3’carbon atom of the sugar and it plays an important role in this process atom (red arrow). In the figure below dideoxythymidine triphosphate — ddTTP, a dideoxynucleotide has been shown that can be added to the growing DNA strand. But the chain elongation ceases when the next nucleotide tries to get attached to the former as there is no 3' -OH for the next nucleotide to be attached to. Hence, the dideoxy method is also called the chain termination method. 

Cell Lysis Procedure

Pellet the cells in a microfuge tube: add 1.5 ml culture, centrifuge for 15 seconds at maximum speed, discard the supernatant, and then repeat the procedure. This will result in the cells from 3 ml of culture in a single tube.

Resuspend the cells in 1 ml Lysis buffer.

Take 300 µl aliquot for Bradford protein assay.

Add 50 µl lysozyme, and mix gently.

After 10-20 minutes at room temperature, microfuge for 10 minutes to pellet cell debris.

Use supernatant for LDH assay.

Principle

Western blot analysis (also called protein immunoblot) is a useful tool for the detection of proteins in biological samples. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose), where they are probed (detected) using antibodies specific to the target protein.  It is a technique for analyzing and identifying protein antigens: the proteins are separated by electrophoresis in polyacrylamide gel, then transferred (“blotted”) onto a nitrocellulose membrane or treated paper, where they bind in the same pattern as they formed in the gel. The antigen is overlaid first with antibody, then with anti-immunoglobulin or protein A labeled with radioisotope, fluorescent dye, or enzyme. 

The method originated from the laboratory of George Stark at Stanford. The name “Western blot” was given to the technique by W. Neal Burnette and is a play on the name “Southern blot”, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting and the detection of posttranslational modification of protein is termed “Eastern blotting”.