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

How to Choose between G250 or R250 Coomassie Dyes

Posted by Protein Man on Apr 22, 2015 10:00:00 AM

11586304016_90f3ded54b_z_1Proteins separated by gel electrophoresis can be visualized using different staining procedures, including Coomassie stains, silver stains and fluorescent stains. Among these staining techniques, silver staining undoubtedly offers the highest sensitivity. However, it also involves a complex and relatively time-consuming protocol, has a low linear dynamic range and reproducibility, and is not compatible with mass spectrometry.

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Dialysis - Knot the Way to Do It

Posted by Protein Man on Apr 17, 2015 10:00:00 AM

Dialysis_-_Knot_the_Way_to_Do_ItThere are certain things that you should not do when performing dialysis in the laboratory. To ensure that all unwanted small molecular weight substances (such as reducing agents, non-reacted crosslinking or labeling reagents, and preservatives) are removed and facilitate a more efficient buffer exchange for macromolecular samples such as proteins, here are some of the things you need to avoid at all cost.   

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Tags: performing dialysis

Why SDS is a Necessary Evil in PAGE

Posted by Protein Man on Apr 2, 2015 9:00:00 AM

6876898938_46a67873fc_z1lSodium dodecyl sulphate-polyacrylamide gel electrophoresis or SDS-PAGE is commonly used in protein aboratories for separating proteins based on their molecular weights. This is accomplished by taking advantage of their differential rates of migration through a sieving matrix (the acrylamide or agarose gel) in response to an applied electrical field.

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Colorimetric or Copper Based Protein Assays - How to Decide Which is Best

Posted by Protein Man on Mar 26, 2015 10:00:00 AM

While protein assays have a variety of uses in life science research, there is no single assay method that is suitable for all applications.  Despite all the advancements in modern science, a protein assay method that is not affected by any non-protein components or by the differences in protein composition does not exist. For this reason, protein laboratories find it necessary to have more than one type of protein assay for research applications.

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Tags: Protein Assays

Why do PVDF membranes require a methanol soak?

Posted by Protein Man on Mar 6, 2015 8:47:18 AM


INTRODUCTION:

PVDF and nitrocellulose membranes are both used in Western blotting and have various different characteristics, however a common question asked is "Why do PVDF membranes require a methanol soak?".

Check out this blog for more on the differences of the membranes:

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Tags: Western Blotting

Which Stain is Most Compatible with Mass Spectrometry?

Posted by Protein Man on Mar 3, 2015 10:00:00 AM

Protein_StainsOnce proteins have been separated and resolved by SDS-PAGE or 2D electrophoresis, the proteins are visualized using different staining procedures. For this purpose, most laboratories worldwide use three common protein staining techniques: Coomassie brilliant blue, silver staining or fluorescent staining. So, how do you know which stain will be best suit for mass spectrometry? Here are some things you need to consider to help you come up with the right decision.

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Tags: Protein Stains

How to Tell if You Should Use Gel Filtration or Dialysis

Posted by Protein Man on Feb 27, 2015 10:00:00 AM

largeBasically, there are two methods that are commonly used in the preparation of protein samples to make them suitable for long-term storage and compatible with downstream applications - dialysis and gel filtration. So, how do you know which one to use? Here are some things you need to consider if you want to increase your chances of making the right decision.

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Tags: Dialysis Efficiency

Chromogenic Protein Detection: Is It a Suitable Alternative to Chemiluminescence?

Posted by Protein Man on Jan 27, 2015 9:00:00 AM

When doing a Western blot procedure, the macromolecules are first separated using gel electrophoresis. The separated macromolecules are transferred onto a second matrix (nitrocellulose or polyvinylidene difluoride (PVDF) membrane) and the membrane is blocked to prevent the nonspecific binding of antibodies to its surface. The transferred protein is then complexed with an enzyme-labeled antibody (probe) and an appropriate substrate is added to produce a detectable product.

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Tags: Protein Detection

How to Improve Dialysis Efficiency

Posted by Protein Man on Jan 21, 2015 8:00:00 AM

In biochemistry, dialysis refers to the process of separating small, unwanted compounds from macromolecules in the sample solution through selective and passive diffusion. Basically, the sample and the buffer solution (dialysate) are placed on opposite sides of the membrane. Since dialysis works by diffusion, molecules will naturally move from areas of higher concentration to areas of lower concentration until the point where equilibrium is reached. This, in turn, will facilitate the separation of molecules in both the sample and the dialysate.

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Tags: Sample Clean Up

The Basics of Hydrophobic Interaction Chromatography

Posted by Protein Man on Dec 23, 2014 2:11:34 PM

Hydrophobic interaction chromatography (HIC) is one of the most widely used methods for separating and purifying proteins in their native state.  HIC also proves to be quite useful in isolating protein complexes and in studying protein folding and unfolding. So, how does HIC work? What are the principles behind this technique? To understand the mechanism behind this type of chromatography, here are some things you definitely need to know.

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Tags: Basics of Hydrophobic

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