Question

Please answer the following questions:

1. Western-blotting: principles and application. Steps involved in western-blotting.

2. Pay attention to following steps: sample treatment (why proteinase inhibitor is added), protein quantification (Bradford method); SDS electrophoresis (separation according to MW).

3. Purpose of blocking? (to prevent non specific antibody binding to the membrane) What are primary antibody and secondary antibody? How to detect antibody-antigen complex?

4. Principles and application of Elisa (disease diagnosis). Steps involved in Elisa. Which enzyme is usually linked? And what is the substrate for the enzyme? What is the final results of Elisa? (color reaction).

5. How to perform fermentation scale up? What is the growth curve? How to choose host system?

Answer 1

Western blot principle

Western blotting is used to visualize proteins which are separated by gel electrophoresis on the basis of their size under the influence of current. The gel is placed next to a nitrocellulose or PVDF (polyvinylidene fluoride) membrane and an electrical current causes the proteins to migrate from the gel to the membrane. The membrane is then be probed by antibodies specific for the target of interest and visualized using secondary antibodies which are HRP-tagged by adding detection reagents.

Sample preparation

Protein is isolated from cell lines or tissue by using ultrasonication or homogenizer or freeze-thaw method. Now the protein concentration is measured by the Bradford method. The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. Serially diluted bovine serum albumin of known concentration is used as a standard. Color is measured in spectrophotometer and protein concentration is measured by obtained optical density.

The sample is prepared by using RIPA buffer (radioimmunoprecipitation buffer), SDS and DTT (to denature the protein), Coomassie brilliant blue (to track the protein run).

Proteinase enzyme is also added to prevent the protein digestion because protein digestion enzymes are present in the sample (lysosomes).

Gel electrophoresis

Polyacrylamide gel is prepared and the sample is loaded in the wells and run in SDS-tris-glycine buffer under influence of current towards the anode (Protein is negatively charged because of SDS).

Transfer of protein to the blotting membrane

The gel is placed on the blotting membrane and transfer under the influence of current in transfer buffer that is tris-glycine.

After the protein transfer, the membrane is blocked by skim milk to prevent the nonspecific binding of antibodies. Every time the membrane is washed with a washing buffer.

Detection of the target protein

Primary antibodies are the antibodies against the target protein, are added to the membrane and kept in cold overnight. Next day the primary Abs are removed, secondary antibodies (Secondary Abs are against the primary Abs which are HRP tagged) are added, kept for two hours.

Now the membrane is used to develop the bands (antigen-antibody complex on the membrane) on the X-ray film in dark by using ECL reagent.

ELISA

ELISA Principle. Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme. ELISAs can provide a useful measurement of antigen or antibody concentration and can be used to detect several diseases.