Abstract
Reversible biotinylation of human C1q without impairment of its physiologic functions has allowed us to develop a simple and rapid purification method for C1q receptor (C1qR). The biotinylating reagent, NHS-SS-biotin (Mr 606.7) contains an extended connector or cross-linker arm which limits steric hindrance and is bridged by a cleavable disulfide bond to the biotin component. Biotinylation was achieved by mixing C1q (in PBS, pH 7.4) with NHS-SS-biotin (dissolved in dimethyl formamide) in a 50:1 v/v and 1:25 mol/mol ratio and allowing the reaction to continue at room temperature for 4 h. The mixture was then dialyzed against PBS pH 7.4 (2 × 1 liter) and analyzed by SDS-PAGE and hemolytic assay using C1q depleted serum. Under these conditions neither denaturation of the protein nor loss of hemolytic activity was evident. Such biotinylated C1q (Bio-C1q) was used to pull out the C1qR from detergent-solubilized (1% NP-40 in PBS, pH 7.4 plus inhibitors) 125I-surface labeled membrane solution that had been first centrifuged (1 h, 45 000 × g, 4°C) and then sequentially precleared with immobilized protein A, protein A-IgG and gelatin. The mixture of Bio-C1q and membrane solution was then incubated (20 h, 4°C), applied to immobilized avidin (equilibrated with PBS, pH 7.4, 0.1% NP-40) and after washing, the bound C1qR was eluted with equilibrating buffer containing 1 M NaCl, and the C1q by same buffer containing 100 mM DTT. The eluted C1qR contained a major Mr 70 000 molecule which upon reduction electrophoresed with an apparent Mr of 85 000-90 000 as assessed by SDS-PAGE analysis. In addition, a faint single chain band of 30-40 kDa was eluted with the major band and may represent a non-covalently associated part of the C1qR molecule.
| Original language | English |
|---|---|
| Pages (from-to) | 251-260 |
| Number of pages | 10 |
| Journal | Journal of Immunological Methods |
| Volume | 110 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jun 13 1988 |
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Keywords
- biotinylation of
- C1q
- C1q ligand
- C1q receptor
- purification of
ASJC Scopus subject areas
- Biotechnology
- Immunology
Cite this
Reversible biotinylation of C1q with a cleavable biotinyl derivative. Application in C1q receptor (C1qR) purification. / Ghebrehiwet, Berhane; Bossone, Steven; Erdei, A.; Reid, Kenneth B M.
In: Journal of Immunological Methods, Vol. 110, No. 2, 13.06.1988, p. 251-260.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Reversible biotinylation of C1q with a cleavable biotinyl derivative. Application in C1q receptor (C1qR) purification
AU - Ghebrehiwet, Berhane
AU - Bossone, Steven
AU - Erdei, A.
AU - Reid, Kenneth B M
PY - 1988/6/13
Y1 - 1988/6/13
N2 - Reversible biotinylation of human C1q without impairment of its physiologic functions has allowed us to develop a simple and rapid purification method for C1q receptor (C1qR). The biotinylating reagent, NHS-SS-biotin (Mr 606.7) contains an extended connector or cross-linker arm which limits steric hindrance and is bridged by a cleavable disulfide bond to the biotin component. Biotinylation was achieved by mixing C1q (in PBS, pH 7.4) with NHS-SS-biotin (dissolved in dimethyl formamide) in a 50:1 v/v and 1:25 mol/mol ratio and allowing the reaction to continue at room temperature for 4 h. The mixture was then dialyzed against PBS pH 7.4 (2 × 1 liter) and analyzed by SDS-PAGE and hemolytic assay using C1q depleted serum. Under these conditions neither denaturation of the protein nor loss of hemolytic activity was evident. Such biotinylated C1q (Bio-C1q) was used to pull out the C1qR from detergent-solubilized (1% NP-40 in PBS, pH 7.4 plus inhibitors) 125I-surface labeled membrane solution that had been first centrifuged (1 h, 45 000 × g, 4°C) and then sequentially precleared with immobilized protein A, protein A-IgG and gelatin. The mixture of Bio-C1q and membrane solution was then incubated (20 h, 4°C), applied to immobilized avidin (equilibrated with PBS, pH 7.4, 0.1% NP-40) and after washing, the bound C1qR was eluted with equilibrating buffer containing 1 M NaCl, and the C1q by same buffer containing 100 mM DTT. The eluted C1qR contained a major Mr 70 000 molecule which upon reduction electrophoresed with an apparent Mr of 85 000-90 000 as assessed by SDS-PAGE analysis. In addition, a faint single chain band of 30-40 kDa was eluted with the major band and may represent a non-covalently associated part of the C1qR molecule.
AB - Reversible biotinylation of human C1q without impairment of its physiologic functions has allowed us to develop a simple and rapid purification method for C1q receptor (C1qR). The biotinylating reagent, NHS-SS-biotin (Mr 606.7) contains an extended connector or cross-linker arm which limits steric hindrance and is bridged by a cleavable disulfide bond to the biotin component. Biotinylation was achieved by mixing C1q (in PBS, pH 7.4) with NHS-SS-biotin (dissolved in dimethyl formamide) in a 50:1 v/v and 1:25 mol/mol ratio and allowing the reaction to continue at room temperature for 4 h. The mixture was then dialyzed against PBS pH 7.4 (2 × 1 liter) and analyzed by SDS-PAGE and hemolytic assay using C1q depleted serum. Under these conditions neither denaturation of the protein nor loss of hemolytic activity was evident. Such biotinylated C1q (Bio-C1q) was used to pull out the C1qR from detergent-solubilized (1% NP-40 in PBS, pH 7.4 plus inhibitors) 125I-surface labeled membrane solution that had been first centrifuged (1 h, 45 000 × g, 4°C) and then sequentially precleared with immobilized protein A, protein A-IgG and gelatin. The mixture of Bio-C1q and membrane solution was then incubated (20 h, 4°C), applied to immobilized avidin (equilibrated with PBS, pH 7.4, 0.1% NP-40) and after washing, the bound C1qR was eluted with equilibrating buffer containing 1 M NaCl, and the C1q by same buffer containing 100 mM DTT. The eluted C1qR contained a major Mr 70 000 molecule which upon reduction electrophoresed with an apparent Mr of 85 000-90 000 as assessed by SDS-PAGE analysis. In addition, a faint single chain band of 30-40 kDa was eluted with the major band and may represent a non-covalently associated part of the C1qR molecule.
KW - biotinylation of
KW - C1q
KW - C1q ligand
KW - C1q receptor
KW - purification of
UR - http://www.scopus.com/inward/record.url?scp=0023937311&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0023937311&partnerID=8YFLogxK
U2 - 10.1016/0022-1759(88)90111-1
DO - 10.1016/0022-1759(88)90111-1
M3 - Article
VL - 110
SP - 251
EP - 260
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 2
ER -