Various techniques designed to separate cell components into subcellular fractions that preserve the structure and physiology of its functioning constituents for biochemical and ultrastructural analysis.
Sigma® cell fractionation assays
Method for detecting a specific protein or message. A spot of solution is dotted onto nitrocellulose paper, a specific antibody or probe is allowed to bind and the presence of bound antibody/probe is then shown by using a coupled secondary antibody, as in immunoblotting or by other visualization methods (fluorescence, colorimetric).
Sigma Dot Blot assays
Competitive ELISAs for the quantitative determination of protein concentration in biological fluids. The specific protein in the sample competes with a fixed amount of the same enzyme-conjugated protein for the constant number of binding sites on the antibody. The intensity of the yellow color is inversely proportional to the concentration of antigen in the standards or samples.
Enzyme-Linked Immunosorbent Assay (ELISA) utilizes an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biological activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed.
- A monoclonal or polyclonal antibody (capture antibody) specific for protein is coated into the wells of a multiwell plate (96 wells in a plate).
- Antigen in standards, unknown samples and controls is added and incubated in order to facilitate binding of antigen and antibody.
- Several washes are performed after each incubation in order to remove excess of unbound reagents.
- A second (detection) antibody specific for the whole protein at specific site is added and incubated 1 hour at RT.
- During the second incubation, this antibody serves as a detection antibody by binding to another epitope of the immobilized antibody.
- After removal of excess detection antibody, HRP-IgG is added and binds to the detection antibody to complete the four-membrane sandwich.
- Substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of antigen present in the original sample.
- The optical density measured at 450 nm in the multiwell plate reader is used to calculate the concentration of protein in question.
Graphic presentation of ELISA
ELISAs for the detection of proteins phosphorylated at a specified residue(s) of serine, tyrosine or threonine.
The format is solid phase sandwich Enzyme Linked-Immuno-Sorbent Assays (ELISA). A monoclonal antibody (capture) specific for a protein (regardless of phosphorylation state) has been coated onto the wells of the microtiter plate. Samples, standards, control specimens and unknowns are incubated for two hours and antigen binds to the capture antibody. An antibody specific for phosphorylated or non-phosphorylated protein (detection) is added and during the second one hour of incubation it binds to the immobilized protein captured during the first incubation. An anti-rabbit IgG-HRP completes the four-member sandwich. The reaction is visualized by TMB substrate solution, which produces color, whose intensity is directly proportional to the concentration of protein present in the original specimen. The reaction is stopped with a stop solution and read at 450 nm in the multiwell reader.
Sigma phosphospecific ELISA assays
Assays in which the protein in question catalyzes a chemical reaction or is generated by an enzymatic reaction that is defined by temperature and kinetic requirements.
Sigma’s enzymatic assays
Flow Cytometry and Fluorescence
Techniques based on the use of fluorescent agents (dyes, probes) during the reaction or as a detection method. Fluorescent agents emit light after excitation by light. The wavelength of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. The fluorescence could be read by flow cytometers (quantitative) or under fluorescent microscopes.
Sigma’s flow cytometry and fluorescent assays
Intracellular distribution of chemicals, reaction sites, enzymes, etc., in living cells and tissues visualized by staining, fluorescence, radioactive isotope uptake, selective metal distribution in electron microscopy, or other methods.
Sigma’s histochemical assays
The precipitation of a multivalent antigen by an antibody, resulting in the formation of a large antigen/antibody complex. The antibody and antigen must be soluble. Precipitation usually occurs when there is near equivalence between antibody and antigen concentrations.
Sigma’s immunoprecipitation assays
Assays which utilize a radioactive tracer attached to a substrate or protein to measure the assay results in gamma or scintillation counters or by autoradiography.
back to top