Hydroxysteroid Dehydrogenase, 11-Beta, Type I (HSD11B2) Enzyme

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. 5-10ul 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.