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

How To Determine Degree of Protein Labeling

Posted by Protein Man on Nov 10, 2015 11:43:21 AM


Knowing the amount of dye conjugated to a protein is essential for predicting the amount of dye required for an experiment and for ensuring good control of fluorescence between experiments.

The following blog post explains how to calculate the degree of protein labeling by a selection of dyes by using protein and dye absorbances.

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Tags: Protein Labeling

How To Elute Tightly Bound Glyoproteins from Concanavalin A (Con A) Agarose

Posted by Protein Man on Nov 5, 2015 1:24:00 PM



Isolation of glycoproteins from protein solutions is routinely performed on concanavalin A (Con A) agarose (or sepharose). Con A is used for the purification of glycoproteins, polysaccharides and glycolipids as it binds molecules containing α-D-mannopyranosyl, α-D-glucopyranosyl and sterically related residues. Con A agarose has also be used in other application areas including purification of enzyme-antibody conjugates, purification of IgM and separation of membrane vesicles.

Con A is a metalloprotein and to maintain its binding characteristics the presence of both Mn2+ and Ca2+ is essential. Each subunit of Con A utilizes one calcium and one manganese ion and these cations can be removed under acidic conditions abolishing the carbohydrate-binding activity.

For the elution of bound molecules the preferred method is to use competitive eluents.  Suitable eluents include, but a re not limited to:

  • methyl-α-D-mannopyranoside [50-200mM]
  • methyl-α-D-glucopyranoside [50-200mM]

Common Elution Techniques are Ineffective!

 Researchers routinely report that they have issues with eluting their protein as seen by lower than expected yields.  This reduced yield also reduces the column capacity.

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Tags: Protein Purification

Monoclonal vs Polyclonal Antibodies

Posted by Protein Man on Nov 4, 2015 10:00:00 AM

While both monoclonal and polyclonal antibodies can be used in a wide variety of applications including Western blot, enzyme-linked immunosorbent assays (ELISA), immunoprecipitation, immunofluorescence, immunocytochemistry, Biochip technology and in the diagnosis of disease, they each have their own advantages which make them useful for different applications. To determine which type of antibodies should be used for a particular application, let us try to understand the difference between the two.

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Tags: Antibody Production

Recombinant Antibodies: An Overview

Posted by Protein Man on Oct 28, 2015 10:00:00 AM

Recombinant antibodies (rAbs) are monoclonal antibodies which are generated in vitro using synthetic genes. Unlike monoclonal antibodies (mAbs) which are produced using traditional hybridoma-based technologies, rAbs do not need hybridomas and animals in the production process.

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Tags: Antibody Production

How to determine reactivity of NHS esters on biotinylation and cross-linking reagents.

Posted by Protein Man on Oct 22, 2015 8:24:23 AM


Many protein modification reagents for biotinylation and cross-linking involve reactive groups that conjugate through primary amines.  Primary amines are found in all proteins and peptides as they make up the N-terminus and are also a component of the lysine residue side chains.  Amines, lysine ε-amines and N-terminal α-amines, are the most abundant group in protein molecules and represent the most common target for biotinylation. For example, BSA contains 59 primary amines, of which up to 35 are available on the surface of the molecules and can be reacted with amine reactive esters.

The most widely used amine reactive biotinylation and cross-linking reagents are the water insoluble N-hydroxysuccinimide (NHS) esters or the water soluble N-hydroxysulfosuccinimide (sulfo-NHS) esters.

The addition of a charged sulfonate (SO3-) on the N-hydroxysuccinimide ring of the sulfo-NHS esters results in their solubility in water (~10mM), but are not permeable to plasma membranes. The solubility and impermeability to plasma membranes makes them ideal for studying cell surface proteins as they will only react with the protein molecules on the outer surface of plasma membranes.

Both forms of the NHS esters hydrolyze rapidly in aqueous solutions (7 hours at pH7, minutes at pH9) and for this reason the NHS esters must be handled and stored appropriately.  The NHS esters should be stored desiccated and by allowed to warm to ambient temperature before opening to avoid condensation.  That being said, repeated opening and closing and inappropriate handling will lead to the introduction of moisture and hydrolysis of the NHS-esters.

Hydrolysis (and conjugation) results in the release of NHS that can be assayed with the following procedure.

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Tags: Protein Labeling, Cross-Linkers

How to Stain Glycoproteins in Polyacrylamide (PAGE) Gels

Posted by Protein Man on Sep 21, 2015 10:00:00 AM

There are a number of staining methods that can be used to detect highly glycosylated proteins on SDS gels, even at very low levels (i.e. up to a few nanograms). Some of the most commonly used stains for this purpose include the Coomassie brilliant blue stain (for proteins with limited glycosylation) and silver stain (used in cases where high sensitivity is required).

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Tags: Protein Detection

Hands-on Science as a Teaching Advantage

Posted by Protein Man on Sep 15, 2015 10:00:00 AM

Most people learn more by doing things rather than by just reading, watching or hearing about it. As such, providing hands-on learning can have a profound effect on learning in schools, particularly on science teaching.

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Why do I need a protease inhibitor?

Posted by Protein Man on Aug 28, 2015 10:00:00 AM

While proteolytic enzymes such as proteases and phosphatases play an important role in living cells and help ensure the survival of the organism, the mechanisms that regulate the tightly controlled cellular environment is disrupted during cell lysis. When this happens, these enzymes may start cleaving a variety of proteins that they would otherwise not touch in intact cells. This situation often leads to reduced recovery of total protein and biologically meaningless representation of protein activities.

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Using Protease Assays for Accurate Protease Detection

Posted by Protein Man on Aug 20, 2015 10:00:00 AM

Proteases are enzymes that facilitate proteolysis, or the breakdown of protein and peptide molecules into smaller polypeptides and/or amino acids. These enzymes do their job by cleaving the peptide bonds linking the amino acids together in the polypeptide chains.

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Tags: Protein Estimation

The Common Failures That can Occur When Using Dialysis Bags

Posted by Protein Man on Aug 11, 2015 10:00:00 AM

Dialysis is a solution-based separation technique used to facilitate the removal of small, unwanted compounds from macromolecules such as proteins, DNA, or polysaccharides through a semi-permeable membrane using the principle of selective diffusion. Although there are a lot of factors that affect the dialysis rate, the semi-permeable membrane is the single most important factor that can determine the success of the process.

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