HSD11B2 enzyme product blog
Tags: Enzyme; Hydroxysteroid Dehydrogenase, 11-beta, Type I; HSD11B2; HSD11B2 enzyme;
The HSD11B2 hsd11b2 (Catalog #MBS653798) is an Enzyme and is intended for research purposes only. The product is available for immediate purchase. MyBioSource\'s Hydroxysteroid Dehydrogenase, 11-beta, Type I can be used in a range of immunoassay formats including, but not limited to, ELISA (EL/EIA) and Antibody blocking. Not suitable for use in Western Blot.Other applications not tested.
ELISA: 50-100ng/well
Antibody Blocking: 5-10ug control peptide per 1ul MBS625504(antiserum)or per 1ug MBS625247 (affintity purified antibody).
Optimal dilutions to be determined by the researcher.
Antibody Blocking -
Principle: It is not uncommon to see more than 1 band in Western Blot when probed with a given antibody or see more diffuse staining in immuno-localization studies. The question arises which one of this band(s)/staining is specific. The antibody specificity is generally studied by competing with excess of antigen (peptide or protein) or immuno-neutralization with the antigens. ln principle, a small volume of antibodv (e.g. 1-5ul) is first reacted with excess peptide (5-50 fold over the antibodv e.g. 1 ug antibody reacted with 5-50ug peptide; exact amounts determined by titration) to neutralize it. The neutralized antibody can no longer subsequently bind to another antigen (a band of interest) or staining pattern. So the band(s)/staining that is competed bv the antigen/peptide is supposedly specific. lf more than one band disappears by peptide/antigen competition then those bands have the antigenic determinants and could be considered either fragments of the large antigen or multimer.
General Protocol for Antibody Blocking:
1. Determine the amount of antibody that is-needed for 1 strip (e.g. 2ml). For example. an antibody has given desired bands at 1:1000 dilution. So you will need 1ul/ml antibody (2ul antibody for 2ml antibody solution). lf an antibody were used at 1:5000 dilution then you would only need 0.2ul/ml (use 2ul of 1:10 dilution for better acturacy).
2. Prepare 2 tubes
2a. Label Tube 1 as "+peptide". Label Tube 2 as "-peptide".
2b. To Tube 1, add antigen/peptide solution (10-50ug peptide or 10-100ul antigen added).
2c. To Tube 2, add same volume PBS (no peptide/antigen) to the other tube. Mix gently.
3. Incubate both tubes at 37�C for 1-2 hrs and 2-24 hrs at 4�C.
4. Centrifuge the tubes for 15 min at 4�C in a microfuge (10-15000rpm) to pellet any immune complexes. Carefully remove the supernatant. lf no visible pellet is seen, then just leave approx. 5-l0ul at the bottom to avoid disturbing invisible immune complexes. If you do not centrifuge the solution, it may give high background.
5. After centrifugation, make up the volume of supernatant to 2ml (or what is necessary depending upon the initial antibody taken) with buffer (PBS, Tween with or without BSA or milk or any buffer that was used initially). Use both antibodies (with and without antigen/peptide) for Western Blot or immuno-localization.
6. Observe the bands/staining that disappear.
Notes:
1. The greater the antibody titer or initial volume of antibody taken, the greater will be the antigen/peptide necessary to completely block the antibody activity.
2. A partial inhibition of antibody activity is an indication that more antigen/peptide will be needed to completely block the antibody.
3. It may be necessary to optimize the amount of antigen/peptides by adding various amounts to a fixed concentration of antibody. Researchers should empirically determine the suitability of the HSD11B2 hsd11b2 for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process.
The HSD11B2 hsd11b2 product has the following accession number(s) (GI #22478066) (NCBI Accession #AAH36780.1) (Uniprot Accession #P80365). Researchers may be interested in using Bioinformatics databases such as those available at The National Center for Biotechnology Information (NCBI) website for more information about accession numbers and the proteins they represent. Even researchers unfamiliar with bioinformatics databases will find the NCBI databases to be quite user friendly and useful.
To buy or view more detailed product information and pricing, please click on the technical datasheet page below:
Synthetic peptide corresponding to 16aa of mouse 11b-HSD1 conjugated to KLH.
11-Beta-hydroxysteroid dehydrogenase (11b-HSD) is a microsomal short chain dehydrogenase/reductase (SDR) which catalyzes the inter-conversion of biologically active glucocorticoid (cortisol in human and corticosterone in rats and mice) and inactive glucocorticoid (cortisone and 11-dehydrocorticosterone). Two tissue specific isoforms (11b-HSD1 and 11b-HSD2) of 11b-HSD with two different functions regarding glucocorticoid availability, have been identified. The decreased 11-beta-hydroxy oxidation of cortisol results in Apparent Mineralocorticoid Excess (AME) disorder which is manifested by hypertension, hypokalemia, low plasma renin activity, and responsiveness to spironolactone. AME is principally a disorder of juveniles and children with this condition oxidize cortisol to cortisone poorly but carry out the reverse process unimpaired. AME arises from mutations in the 11-beta-HSD2 gene. The glucocorticoids can be produced locally by 11beta-HSD1 and increased visceral accumulation of glucocorticoids results in visceral obesity, insulin resistant diabetes, hyperlipidemia and hyperphagia.
11betaHSD-1 (variously termed as HSD11L; mouse, 292 aa, rat 287 aa, human 292 aa) is a ~35kD glycosylated membrane-protein, oriented into the lumen of endoplasmic reticulum. This isoform is the sole 11b-reductase in the body and exerts two separate enzymatic activities: 11-beta-dehydrogenase (cortisol to cortisone) and 11-oxoreductase (cortisone to cortisol) in vitro. In vivo, it acts mainly as reductase producing active cortisol. The enzyme also plays an important role in xenobiotic carbonyl compound detoxification processes. 11b-HSD1 is expressed in a wide array of tissues, with highest level in Liver and adipose tissues. The increased adipocyte 11b-HSD1 is a common mechanism for visceral obesity and metabolic syndrome. Although the deficiency in 11b-HSD1 activity is not related to AME, it results in a syndrome characterized by an increased adrenocorticotropic hormone (ACTH)-driven androgen production. In mouse, the over-all aa sequence of 11b-HSD1 is approximately 18% identical to that of 11b-HSD2.