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

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

Membrane Protein Isolation and Purification With Phase Separation

Posted by Protein Man on Dec 22, 2014 11:00:00 AM

It is interesting to note that while detergent-solubilized membrane proteins can be purified and concentrated through phase separation (which is a simple, efficient and cost-effective method that can be used for this purpose), most membrane protein scientists do not use this method or even know that such methods exist. To better understand and appreciate how this method works, here are some things you definitely need to know.

Phase Separation: Understanding the Basics

In a nutshell, phase separation (also known as cloud point extraction) refers to the state wherein the micelles in an aqueous solution become immiscible with water and form large aggregates that will separate from the water phase. This condition is usually achieved upon the addition of detergents or when the temperature or salt concentration of the solution is altered.

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Tags: Protein Purification

Magnetic Beads for Immunoprecipitation

Posted by Protein Man on Nov 18, 2014 9:00:00 AM

Researchers have traditionally used agarose beads for immunoprecipitation (IP). However, there has been a growing trend in recent years favoring the use of magnetic beads. According to a recent survey of 1,013 scientists, 60% of the respondents will start using magnetic technology in the next three years.

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Tags: Protein Concentration, Antibody Production, Protein Extraction

PVDF or Nitrocellulose - Which Membrane is Best?

Posted by Protein Man on Nov 12, 2014 9:00:00 AM

When it comes to Western blotting, choosing the right membrane for your application usually spells the difference between success and failure. However, this is usually easier said than done since you need to consider the properties of your protein and the downstream detection steps required in your application before you can determine which membrane can give you the results that you need.

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Tags: Western Blotting

A Look into the 6XHis Tag and its Uses

Posted by Protein Man on Oct 20, 2014 9:03:00 AM

The 6xHis tag, also known as polyhistidine tag, His6 tag and/or hexa histidine tag, is an amino acid motif consisting of at least 6 histidine residues fused to the carboxyl (C-) or amino (N-) terminus of a target protein in transfected cells. This tag is most commonly used in the production of recombinant proteins since the string of histidine residues binds to several types of immobilized ions (such as nickel, cobalt and copper) under specific buffer conditions to allow for the simple detection and purification of His-tagged proteins.

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Tags: Protein Purification

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