There are a wide variety of ELISA substrates designed for different systems and detection methods. These ELISA substrates are designed to detect alkaline phosphatase (AP) and horseradish peroxidase (HRP). In addition to their sensitivity, other factors, such as cost and ease of use, may also factor in. Choosing the right ELISA substrates begins with determining the limits of detection required. From there, a general type of ELISA substrate can be selected and the specific substrate can be narrowed down. There are five major types of substrate available: PNPP, ABTS, OPD, TMB, and ONGP. Of these, TMB is one of the most versatile.
The Protein Man's Blog | A Discussion of Protein Research
Cells are made up of recognizable structures that each serve their own purposes and have their own characteristics. Common subcellular markers are often used to distinguish cells from each other and to identify potential irregularities within the cellular structure. Though there are a multitude of subcellular markers available, a few of them are more common and more easily identified than others.
Extraction of proteins from cells and tissue of organisms is the first step towards isolation of proteins. The extracellular matrix needs to be removed or digested in case of tissue, the cell wall needs to be digested for organisms like bacteria, yeast and plants, and the cell membrane needs to be disrupted to release the proteins in solution. Traditionally, physical methods for disruption of cells and tissues are employed to release cellular proteins including sonication, french press, homogenization, manual grinding or using blenders. Although one is able to get the active proteins, these physical methods have several limitations:
Protein analysis and identification through mass spectrometry first requires a breakdown of each protein into their composite peptides. Once the protein has been broken down, the peptides can be separated through the use of a reverse phase column and the peptides and peptide fragments can be measured using a mass spectrometer.
Tags: Mass Spectrometry
Adjuvants play an important role in the antibody production by acting as immunopotentiators. They augment immune response via different mechanisms depending upon the adjuvant such as ‘depot’ effect, antigen presentation, antigen targeting, immune activation or modulation and cell-mediated response. The goal of adjuvant for antibody production is that high affinity, high titer and high avidity (for polyclonal) antibodies are raised. Both humoral and cell mediated response are necessary to achieve efficient antibody production. There are hundreds of preparations for adjuvants described in literature depending upon specific needs of an investigator. Nevertheless, most commonly and widely used adjuvants for antibody productions are few as listed below.
Tags: Antibody Production
Immunodetection of proteins on a Western blot requires the use of expensive antibodies, but poor or incomplete transfer would result in wasting these antibodies. A Western transferred blot can be checked for success of protein transfer with a protein stain and therefore prevent wasting expensive antibodies. Various options for protein stains for Western blot are available, including, but not limited to, Ponceau S, Amido black 10B, Coomassie brilliant blue R250, India ink or colloidal gold. In addition to checking success and quality of Western transfer, staining blots is also used for semi-quantitative protein estimation. In addition, checking the blot with a stain gives a rough idea to a researcher that the desired protein is present (based on size, mobility etc) and whether to go for immunodetection especially when expensive and limiting amounts of antibodies are available.
Tags: Western Blotting
How to visualize proteins after electrophoresisIn-gel visualization of proteins separated by SDS-PAGE or by 2-D gel electrophoresis is achieved by staining gels with dye, metals ions or fluorescent stains. Different staining method are preferred depending upon broad range binding of stain to proteins, the sensitivity to detect low abundant proteins and downstream application desired for a separated protein like mass spectrometry. Sometimes two staining methods are carried out as single staining method cannot achieve everything desired by a researcher. Various staining method for in-gel visualization of proteins are as follows.
Coomassie Brilliant Blue stainingThe Coomassie dyes R-250 and G-250 bind to proteins stoichiometrically through their sulfonic acid groups. The interactions between dye and protein are Van der Waals and ionic. The sulfonic acid groups interact with positive amine groups. Therefore coomassie dye binds to wide range of proteins. The most commonly used dye R-250 can detect protein as low as 0.1 µg. Coomassie Brilliant Blue involves staining and destaining step as the gel also turns blue. Coomassie Brilliant blue is compatible with mass spectrometry analysis. Though widely used for staining gels to view proteins, its not a method of choice if higher sensitivity is desired. For analytical purposes other methods like silver staining and fluorescent staining are preferred over classical Coomassie Brilliant Blue staining.
Colloidal Coomassie G-250 stainingIn colloidal Coomassie staining G-250 dye is used instead of R-250 due to its colloidal properties. G-250 forms colloidal particles in high alcoholic solutions containing strong acids and high concentration of salts 1, 2. Formation of colloidal G-250 particles reduces free dye in solution and thus the dye does not penetrate and stain gel. This step enhances the sensitivity of staining proteins and removes background staining of gels. With colloidal Coomassie staining detection limit of 1-10 ng of protein in gel is achieved 3.
Silver stainingIt offers high sensitivity of less than 1 ng of protein 4. The silver ions bind to the protein and are reduced further with a developing solution to give an image of protein bands. Silver staining involves various steps like protein fixation, sensitization, silver impregnation and image development. Based on solution used there are two methods of silver staining: silver nitrate and silver-ammonia complex method. Silver staining protocols compatible with downstream application like mass spectrometry are available. The downside of silver staining is that it has limited dynamic range and thus do not interact uniformly with all proteins. In addition it involves use of hazardous chemicals.
Fluorescent stainingFluorescent staining uniformly stains protein, is rapid and offers sensitivity equal to that attained by silver staining method. It involves no destaining step and is compatible with mass spectrometry and microsequencing. Commercially fluorescent stains are available to stain 1D or 2D gels to visualize bands.
Stain free detection of proteins in gelIn stain free method for visualizing proteins, 2, 2, 2-Trichloroethnaol is added to the polyacrylamide solution before casting a gel 5. The principle behind stain free method is that the tryptophan amino acids in protein undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in visible range (300 nm). This method is fast as one can see bands on transilluminator within 5 minutes and it avoids use of stains.
References1. Neuhoff, V. et al (1988). Electrophoresis 9, 255-262
2. Candiano, G. et al (2004). Electrophoresis 25, 1327-1333
3. Kang, D. et al (2002). Bull. Korean Chem. Soc. 11, 1511-1512
4. Chevallet, M. et al (2006). Nat Protoc.1(4): 1852-1858
5. Ladner, C. L. et al (2004). Anal Biochem. 2004 Mar 1, 326(1):13-20
Tags: Protein Detection
Crosslinking reagents or crosslinkers are used to covalently bind two or more protein molecules to facilitate the identification of relationships between near-neighbor proteins, ligand-receptor interactions, three-dimensional protein structures, and molecular associations in cell membranes. In the same manner, they can also be used to modify nucleic acids, drugs, and solid surfaces, and in the preparation of antibody-enzyme conjugates and immunotoxins.
Protein solubility is a key factor for successful 2D gel electrophoresis. Thus, the choice of protein solubilization buffers can greatly affect the results of your experiments. Proteins need to be solubilized during sample preparation and electrophoresis to break the inter- and intra-molecular interactions involved in protein aggregation (e.g. disulfide and/or hydrogen bonds, ionic and/or hydrophobic interactions, and van der Waals forces). Failure to break these interactions may result in sample loss or the formation of experimental artifacts.
When treated with a cytotoxic compound, living cells may face one of two fates. They could either stop growing and dividing, or die through either of two distinct processes - necrosis or apoptosis. Basically, cells undergoing necrosis (accidental cell death) swell and lose membrane integrity before shutting down and releasing their intracellular contents into the surrounding environment. This type of cell death is usually triggered by external factors such as toxic chemical or traumatic physical events.
Tags: Cytotoxicity Assays