In any Synthetic Biology experiment which involves working with proteins, the first step is quantifying the amount of protein present in the sample - irrespective of which protein it is. Before running an [SDS-PAGE](#), for instance, we must know the concentration of the protein so that we can appropriately decide how much to load. This is done using **Bradford's Assay.**
When light passes through a sample, the components of the sample absorb some of the light. Bradford's Assay is a colourimetric assay, based on **Beer-Lambert's Law** - which says that the absorbance (amount of light absorbed as a fraction of the total light incident) of a particular solution is directly proportional to the concentration of the solution.
Bradford's Assay makes use of the dye CBB G-250 (Coomassie Brilliant Blue). On binding to a protein, the absorbance maximum (wavelength of light absorbed) shifts from 465nm to 595nm. This is because:
1. CBB donates free electrons to ionisable groups on protein, which disrupts the native state (the active, folded form of the protein) and exposes hydrophobic pockets
2. Hydrophobic pockets bind to the non polar region of the dye via van der Waals forces. As a consequence, positive amine groups in the protein come tête-à-tête with the negative charge of CBB.
3. This ionic interaction stabilises the complex
Thus, absorbance is measured at 595nm, and the absorbance is proportional to the amount of bound dye. Using a standard curve of known protein concentrations vs the absorbance, we can find the protein concentration in an unknown sample. This is the utility of Bradford's Assay.
#### References:
1. Kielkopf, C. L., Bauer, W., & Urbatsch, I. L. (2020). Bradford Assay for Determining Protein Concentration. Cold Spring Harbor protocols, 2020(4), 102269.
**Western Blotting** is an essential step in protein purification. It involves the transfer of proteins from a gel medium to a membrane. [SDS-PAGE](#) precedes Western Blotting, and is used to separate proteins in a sample based on their molecular weight, under the influence of an electric field parallel to the gel surface. In contrast, Western Blotting is the transfer of separated/resolved proteins (by SDS-PAGE) to a membrane, by an electric field perpendicular to gel surface - causing proteins to move out into the membrane.
Why is Western Blotting important? It helps in the detection specific proteins in a blood or tissue sample. THis is known as **probing.** It is a fundamental step in the separation and identification of proteins in confirmatory medical diagnoses (such as HIV and Lyme Disease), as well as for protein localisation in cells. it is a more specific technique than ELISA (Enzyme-Linked Immunosorbent Assay).
Interestingly, the name Western Blotting comes from a similar technique used for the transfer of DNA strands - called Southern Blotting after the scientist Robert Southern. The analogous techniques for RNA and proteins were simply named Northern and Western - and not because they were after scientists by those names!
The membrane (on to which proteins are transferred) is between the gel surface and positive electrode in a sandwich. After SDS-PAGE, all the proteins have already acquired a uniform negative charge. A fiber pad or sponge is kept at positive end (the top) and filter papers are used to protect the gel and blotting membrane.
Two things are important here - close contact of the gel and the membrane (to get a clear image), and the correct placement and orientation of the membrane between the gel and the positive electrode.
Western Blotting is followed up with **[immunostaining](#)** to identify where the protein of interest is. In simple words, it makes use of the specificity and fidelity of antigen-antibody interactions. An antibody specific to the protein of interest is made to bind with it, and a secondary antibody binds to the primary antibody and either gives of a fluorescent or radioactive signal (**autoradiography**) or is conjugated with an enzyme which can be quantified using an assay. For instance, the enzyme horseradish peroxidase acts on substrate TMB in the presence of hydrogen peroxide to give a bright blue oxidised product.
The membrane used in Western Blotting is broadly of two types:
1.**PVDF (Polyvinylidene Difluoride):** has better mechanical support, allows reprobing and storage, but background noise is higher which necessitates good washing
2.**Nitrocellulose (used in Southern Blotting too):** has high affinity to protein, high retention ability, but brittle, and doesn’t allow reprobing
**Reprobing** is the process of removing primary and secondary antibodies used for immunostaining and carrying out immunostaining again with different antibodies to detect additional proteins of interest.
**Blocking** is an essential step in Western Blotting and immunostaining. It blocks the sites on the membrane not occupied by proteins, to prevent non-specific antibody binding. This is usually done with 5% BSA (bovine serum albumin, a protein) or casein (a milk protein)
#### References:
1. Mahmood, T., & Yang, P. C. (2012). Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 4(9), 429–434.
