Complement Factor Bb antibody

Catalog No. ABIN2473066

Product Details anti-Complement Factor Bb Antibody

Target: Complement Factor Bb

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This Complement Factor Bb antibody is un-conjugated

Application: Flow Cytometry (FACS), ELISA, Western Blotting (WB)

Specificity: Specificity property: neoantigen
The monoclonal antibody, clone 032B-22.1.X, binds to a neoantigen expressed on the Bb fragment of human Factor B. This antigen is not expressed on native Factor B and, accordingly, the antibody does not bind to native Factor B found in human plasma.

Characteristics: Purified IgG

Purification: Purified

Clone: 032B-22-1-X

Isotype: IgG2a

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

If the Factor B is denatured in any way, the protein can express neoantigen(s) that are not present in functionally active, native Factor B. For example, if the Factor B is bound to a well in a microtiter plate, a portion of it will be conformationally distorted and express the Bb neoantigen. Purification of Factor B can also result in denaturation of a portion of the Factor B and this denatured protein can also express the Bb-specific neoantigen. Finally, lack of careful handling of serum and plasma can result in complement activation and generation of small amounts of Bb and Ba fragments; in this case low levels of Bb will be detectable with this antibody and with the MicroVue assay for Bb.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #103567 (Western Blotting)

by: Complement Physiopathology, Departamento de Microbiología I, Universidad Complutense de Madrid

No.: #103567

Date: 02/22/2019

Antigen: Bb

Lot Number: 146096

Method validated: Western Blotting

Positive Control:

Purified Bb fragment and zymosan activated serum

Negative Control:

Non-activated sera

Notes:

ABIN2473066 recognizes both Factor B and Bb fragment in plasma samples. It does not specifically recognize a neo-epitope in Bb fragment.

Validation Images

Western blot analysis of purified Bb fragment (1), zymosan activated serum (2), and non-activated sera (3, 4, 5) using ABIN5692886.

Full Methods

Primary Antibody: ABIN2473066

Secondary Antibody: goat anti-mouse Immunoglobulins/HRP (Dako, P0447, lot 20058518)

Full Protocol:

• Purify Bb fragment from plasma samples.
• Separate 10ng purified Bb fragment and 0.5µl of zymosan activated serum and 0,5 µl of control serums on a 10% SDS-PAGE electrophoresis gel for 1h 120 V.
• Activate PVDF membrane with methanol for 15sec and rinse with transfer buffer for 5min. Transfer proteins from the gel to the PVDF membrane using a Mini Trans-Blot cell Tetra Blotting Module (Bio-Rad, 1660827EDU) for 1h at 100V.
• Stain membrane using Ponceau S to verify protein transfer.
• Block the membrane with blocking buffer (1x PBS, 3% BSA, 0.1% Tween) for 30min at RT.
• Incubate membrane with primary mouse anti-complement factor Bb antibody (antibodies-online, ABIN5692886, lot 146096) diluted 1:1000 in working buffer (1x PBS 1% BSA, 0.1% Tween) ON at 4°C.
• Wash membrane 3x for 5min with PBST (1x PBS, 0.1% Tween).
• Incubate membrane with secondary goat anti-mouse Immunoglobulins/HRP (Dako, P0447, lot 20058518) diluted 1:1000 in working buffer for 30min at RT.
• Wash membrane 3x for 5min with PBST.
• Reveal protein bands using ClarityTM Western ECL substrate (Bio-Rad, 170-5061, lot 102031078) on a ChemiDoc MP Imaging System (Bio-Rad, 17001402).

Experimental Notes:

• In dentatured samples the complement Factor Bb antibody directed against a neo-epitope in Bb fragment ABIN2473066 reveals two proteins of the expected molecular weight of Factor B (90kDa) and Bb fragment (60kDa). The antibody does not specifically recognize a neo-epitope in Bb fragment and is thus not suitable for selectively detecting the Bb fragment subsequently to denaturation.

• As an additional control we performed a Western Blot using in-house antibodies which detect Factor B and fragments Bb and Ba (not shown).

Handling

Format: Liquid

Concentration: 1.0 mg/mL

References for anti-Complement Factor Bb antibody (ABIN2473066)

Xu, Narayana, Volanakis: "Structural biology of the alternative pathway convertase." in: Immunological reviews, Vol. 180, pp. 123-35, (2001) (PubMed).

Target Details for Complement Factor Bb

Target: Complement Factor Bb

Abstract: Complement Factor Bb Products

Gene ID: 629

UniProt: P00751