From bb7f6c1242544a2f0af3aabefef8ee947f53dec6 Mon Sep 17 00:00:00 2001
From: Avani Sardana <avanisardana@MacBookAir.local>
Date: Wed, 2 Oct 2024 16:04:21 +0530
Subject: [PATCH] new blogposts

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 wiki/blogposts/bradford.md    | 19 +++++++++++++++++++
 wiki/blogposts/westernblot.md | 29 +++++++++++++++++++++++++++++
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+---
+title: Bradford's Assay
+author: Parth Kumar
+date: 28 August 2024
+---
+
+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.
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+---
+title: Western Blotting
+author: Parth Kumar
+date: 5 September 2024
+---
+
+**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.
+
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