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

How (and When) to use Viability Assays

Posted by Colin Heath 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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Tags: 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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Tags: 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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How to Ensure Proper Protein Transfer to a Membrane

Posted by Protein Man on Aug 23, 2016 10:00:00 AM

Poor protein transfer can lead to either a weak signal or a lack of signal being observed during the process of western blotting. Though poor protein transfer does occur fairly often, it's also commonly disregarded as a potential cause for a failure. Consequently, it's an issue that should be considered first when trying to determine the cause of a poor signal. 

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Role of Lytic Enzymes in Protein and DNA Extraction

Posted by Protein Man on Aug 16, 2016 10:00:00 AM

Lytic enzymes have a fairly prominent role in protein and DNA extraction. There are multiple enzymes that can be potentially used for lysis, depending on the applications and your lab's unique needs. Understanding lytic enzymes is essential to the process of successful protein extraction. Though physical lysis has been used in the past, it's no longer feasible -- and it's always important that lysis be completed with the proper lytic enzymes, to yield the most valuable results. 

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