Complement Factor Bb anticorps

Détails pour le produit réf. ABIN2473066
Antigène
Reactivité
Humain
7
Hôte
Souris
6
1
Clonalité (Clone)
Monoclonal ()
Conjugué
Inconjugué
1
1
1
1
1
Application
Flow Cytometry (FACS), ELISA, Western Blotting (WB)
5
3
3
3
2
2
1
1
Options
'Independent Validation' signe
Antigène Bb
Numéro du lot 146096
Application validée Western Blotting
Contrôle positif

Purified Bb fragment and zymosan activated serum

Contrôle négative

Non-activated sera

Conclusion

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

Anticorps primaire ABIN2473066
Anticorps secondaire goat anti-mouse Immunoglobulins/HRP (Dako, P0447, lot 20058518)
Protocole
  • 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).
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).

Validation Images
Western Blotting validation image for anti-Complement Factor Bb antibody (ABIN2473066) Western blot analysis of purified Bb fragment (1), zymosan activated serum (2), and n...
Clone 032B-22-1-X
Isotype IgG2a
Specificité 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.
Attributs du produit Purified IgG
Purification Purified
Antigène
ID gène 629
UniProt P00751
Indications d'application Optimal working dilution should be determined by the investigator.
Commentaires

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
Format Liquid
Concentration 1.0 mg/mL
Produit citée dans: Xu, Narayana, Volanakis: "Structural biology of the alternative pathway convertase." dans: Immunological reviews, Vol. 180, pp. 123-35, 2001 (PubMed).

Background publications Sulser: "[The rhabdomyosarcoma with regard to age, sex, localization, pathological anatomy, and prognosis (author's transl)]." dans: Virchows Archiv. A, Pathological anatomy and histology, Vol. 379, Issue 1, pp. 35-71, 1978 (PubMed).

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