The Protein Man's Blog | A Discussion of Protein Research

Best Blocking Buffer Selection for ELISA & Western Blot

Posted by Protein Man on Jan 10, 2017 8:02:17 AM

Antibodies are used to detect specific proteins during research. There are a variety of tests including the Western blot, that produce a surface area that can contain other proteins and components that need to be blocked. Blocking buffers are used to bind the surface areas that may attach to reagents during the testing process, preventing the non-specific antibodies from interacting with the reagents and causing a poor reading or inaccurate results. Knowing which buffers to use, however, is critical for ensuring the accuracy of a test, as each option has its own advantages and disadvantages.

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Topics: Western Blotting, Assay Development (ELISA), Protein Detection

Polyclonal Antibody: Advantage over Monoclonals?

Posted by Protein Man on Jan 6, 2017 8:00:46 AM

Polyclonal antibodies are groups of antibodies that are able to identify many different epitopes of a particular antigen. The antibodies are derived from a selection of B cells, each one recognizing one particular epitope. A monoclonal antibody has one single source. Polyclonal antibodies are easy to store, bind much more quickly to the antigen in question, and provide more powerful protection when it comes to identifying and dealing with specific epitopes.

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Topics: Antibody Production

Viability Assays: Different Types and Their Use in Cell Death Research

Posted by Protein Man on Nov 22, 2016 8:00:00 AM

Viability assays are strategies and techniques that are generally used to determine whether or not an organ, cell, or tissue remains viable or could potentially recover its viability. In general, viability assays will look at the cells or tissue involved to see if they still react and move as they are supposed to. Mitotic activity, mechanical activity, and motility are all used throughout a viability assay for this reason.

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What is Protein Electrophoresis and When Should It Be Used?

Posted by Colin Heath on Nov 15, 2016 8:00:00 AM

When proteins need to be segregated out from a sample, protein electrophoresis may be used. Protein electrophoresis utilizes a matrix and an electrical current in order to easily separate proteins without disruption. Protein electrophoresis is beneficial for a few reasons: it can be performed on a relatively small sample size, it is accurate, and it is simple. There are a few different methods of protein electrophoresis, which may or may not involve a denaturing gel.

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A Derivatizing Agent for Glutathione Assays

Posted by Protein Man on Nov 11, 2016 11:00:00 AM

4-Vinylpyridine as a derivatizing agent for free thiols and its application in Glutathione assay

Glutathione assay involves quantification of reduced Glutathione (GSH) and Oxidized Glutathione (GSSG) in various organisms or in various tissues, blood samples, plasma, serum or cultured cells. Free thiols such as GSH can be detected by their property of relatively high reactivity compared to other biological molecules. On contrary disulfides such as GSSG does not have any unique property that could be exploited for its quantification. Hence Glutathione disulfide (GSSG) quantification can be done after reducing it to its corresponding thiol (GSH). Thus the most widely adopted methodology for quantification of GSH and GSSG is determination of total GSH concentration, followed by alkylation to remove GSH and then reduction of GSSG and its quantification. GSH concentration in sample can be determined by subtracting glutathione disulfide concentration from total glutathione concentration.  

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Topics: Apoptosis Assays

Glutathione Assays To Monitor Oxidative Stress

Posted by Protein Man on Nov 10, 2016 9:53:28 AM

Monitor oxidative stress to determine the extent of cell damage using a colorimetric gluthathione assay.

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Topics: Cytotoxicity Assays

The Advantages of Loading Controls in Western Blotting

Posted by Protein Man on Oct 18, 2016 8:00:00 AM

Loading controls serve as a vital method of verifying the results acquired through western blotting. Loading controls are antibodies that are used to detect proteins within samples. When western blots are used to determine the levels of protein expression in a sample, loading controls ensure that the results aren't due to loading or protein transfer errors. When using a loading control, the right type of control must first be identified. 

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Tips for Preventing a High Background During Western Blotting

Posted by Protein Man on Oct 12, 2016 8:00:00 AM

A high background is one of the most common quality control issues encountered during the process of western blotting. When a high background occurs, it can be very difficult to distinguish the relevant data from the irrelevant data. A uniform high background can be caused by numerous issues and may take some work to resolve -- but without resolving the situation, the blot may not be readable.

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The Top Methods for DNA Denaturation

Posted by Protein Man on Sep 19, 2016 10:00:00 AM

DNA denaturation is the process of breaking down the DNA molecule, generally for the purposes of comparison or sequencing. As with many laboratory techniques, there are a variety of ways to denature DNA -- and each of them tend to be better for specific applications. The top three methods of DNA denaturation are heat, NaOH treatment, and salt. Each of these methods will break the bonds between strands, but may do so with a greater degree of accuracy or lessened disruption.

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What is Drop Dialysis: An Overview

Posted by Protein Man on Sep 12, 2016 10:00:00 AM

After the preparation of DNA solutions, some of the enzymes involved may be impacted by the residues of the chemicals that have been used. This may include excess salt, SDS, or other inhibitory substances. In order to properly utilize the results of the DNA preparation, it is sometimes required to perform a drop dialysis. Ideally, the drop dialysis will be able to "wash" the enzymes in question, removing the residue and providing a better and more accurate result.

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