When doing biological applications in the laboratory, it is essential that you have your phosphate buffers available at all times. This is of extreme importance since most biological applications are very sensitive to changes in pH, and these buffers are very effective in keeping the pH range of cellular fluids within the normal range (6.9 to 7.4).
The Protein Man's Blog | A Discussion of Protein Research
The use of peptides for the generation of antibodies against specific peptides has become an essential tool in proteomic research. However, while they are designed to be good epitopes by themselves, these immunogenic peptides or haptens are too small (size ranges from 1000-2000 Daltons) to elicit a strong antibody response, even when emulsified in an appropriate adjuvant.
The ability to accurately quantify protein concentration is the key to a successful laboratory workflow, and is often required prior to processing protein samples for isolation, separation, and analysis. To determine the total protein concentration in a sample, one of the first factors to consider is the selection of an appropriate protein assay method. However, since there are a wide variety of protein assays available, you need to take a number of factors (e.g. the accuracy required and the amount and purity of the protein available) into account to make sure that you are using the most suitable assay for your application.
Tags: Protein Detection
The serine protease trypsin (Tn) is commonly used in most proteomics experiments to digest proteins into peptides which can be analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS).
When you lyse cells, either through physical disruption or by adding detergents, you also release a variety of other proteins (proteases) that are potentially harmful to your target protein. These proteases can cleave the peptide bonds between the amino acids of polypeptide chains and leave you with multiple short peptide fragments. When this happens, you will end up getting erroneous results from your experiments (western blotting, reporter gene analysis, protein interaction or activity assays).
Tags: Protease Inhibitors
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.
Tags: Protein Labeling
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.
Tags: Protein Purification
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.
Tags: Antibody Production
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.
Tags: Antibody Production
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.