The Blotting Series: Western Blotting

#WesternBlotting is the most widely used of all the Blotting #techniques till date and is an excellent tool to detect and identify specific proteins from protein pool often obtained from #CellLysate. Lets dive deeper to understand the nitty-gritties!

Protocol

1. Electrophoresis: The protocol involves running the protein samples on #PolyacrylamideGel. This could be #SDSPAGE or #NativePAGE until the marker fully resolves.

2. Blotting: Next is transfer of the separated proteins from gel onto a sheet of #NitrocelluloseMembrane. This method is termed as #WesternBlotting with analogy to #SouthernBlotting. The transfer is achieved in a process called #Electroblotting.Here a sandwich of the gel and nitrocellulose membrane is compressed in a cassette, immersed in a #TransferBuffer and put between two parallel electrodes. The current passes at right angles to the gel and this causes the protein bands to electrophorese out of the gel and stick to the membrane as #Blots.

3. Blocking: The blot is then immersed in 10% w/v #BSA or 5% w/v #SkimmedMilk for blocking all remaining #Hydrophobic binding sites on the #NitrocelluloseMembrane. This is the so called #BLOTTOTechnique (Bovine Lacto Transfer Technique Optimizer).

4. Antibody binding: The blot is then incubated in ~1:1000 diluted primary #antibody targeted against the protein of interest. The IgG molecules has their variable region complementary to any region of the target protein. A washing step is done.

5. Enzyme-labelled Antibody tagging: Following the overnight primary antibody treatment, the blot is then incubated with an enzyme-labelled secondary #antibody raised in a different host animal targeted towards the constant region of the primary antibody. A washing step is done.

6. Detection: Upon binding with the secondary antibody, the blot is immersed in the enzyme-#substrate. Upon reaction an insoluble product is precipitated on the membrane at the position of the target #protein. This makes detection easier.

Merits

1. Easy and #specific detection of proteins on the basis of two-factor #authentication with primary and secondary antibody.

2. #Sensitive method

3. No use of #Radioactive probes or toxic stains.

Demerits

1. Lengthy procedure involving at least a whole day (8 hours).

2. Use of SDS-PAGE gel demands use toxic materials like #APS and #TEMED.

3. Requires special imaging apparatus and software for analysis.

Applications

1. Detection of target proteins in a cell lysate sample

2. Determination of gene expression level of a target protein in a cell, by #densitometric analysis of blot bands in control and treated cells.

#HailtotheGoodGenes

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