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Complement Technology Inc human complement proteins c3, factor b and factor d
Human Complement Proteins C3, Factor B And Factor D, supplied by Complement Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human complement proteins c3, factor b and factor d - by Bioz Stars, 2026-08

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C3b binds more avidly to denatured plasma proteins. Panel A: Fluorescence emitted by TNS binding (Ex/Em 322/465 nm) to native or guanidine hydrochloride (GuHCl) denatured DBP to measure the level of protein denaturation. Panel B: Native DBP or GuHCl DBP (both at 0.4 mg/ml) were allowed to form complexes with either 1.3 mg/ml C3 (physiological concentration), 0.26 mg/ml C3 or no C3 in the presence of factor B (fB, 0.2 mg/ml), factor D (fD, 1 μg/ml) and 0.5 mM Mg2+. All proteins were mixed at physiological (plasma) ratios with the exception of reduced or no C3 samples. The samples were incubated at 37°C for 15 minutes. Aliquots were analyzed using SDS-PAGE with β-mercaptoethanol on an 8% gel and then immunoblotted for DBP and C3 (for complex formation) and factor B (for complement activation). Panel C: Elastase inhibitory activity of α1PI was tested using the chromogenic substrate AAPV-pNA by measuring the release of pNA at 405 nm. Native and GuHCl denatured α1PI (both at 60 nM) were incubated at 37°C for 30 minutes with purified neutrophil elastase (60 nM), enzyme activity was then tested by adding 100 μM AAPV-pNA in assay buffer (0.1 M HEPES, pH 7.25 with 0.5 M NaCl, 0.05% Tween 20) and incubated at 37°C for another 30 minutes. Negative control is substrate alone without elastase, positive control is substrate and elastase only. AAPV-CMK sample is elastase pretreated with this peptide inhibitor as an additional control for inhibition of enzyme activity. Panel D: Native or GuHCl α1PI (both at 1 mg/ml) were allowed to form complexes with either 1.3 mg/ml C3, 0.26 mg/ml C3 or no C3 in the presence of factor B (fB, 0.2 mg/ml), factor D (fD, 1 μg/ml) and 0.5 mM Mg2+. All proteins were mixed at physiological (plasma) ratios with the exception of reduced or no C3 samples. The samples were incubated at 37°C for 15 minutes. Aliquots were analyzed using SDS-PAGE with β-mercaptoethanol on an 8% gel and then immunoblotted for α1PI and C3 and factor B. Panel E: Densitometry of the C3b:DBP complex bands in panel B (DBP blot) and C3b:α1PI complex bands in panel D (α1PI blot) comparing native versus GuHCl denatured proteins with the physiological (1.3 mg/ml) C3 concentration.

Journal: Molecular immunology

Article Title: Enhanced recognition of plasma proteins in a non-native state by complement C3b. A possible clearance mechanism for damaged proteins in blood

doi: 10.1016/j.molimm.2014.10.022

Figure Lengend Snippet: C3b binds more avidly to denatured plasma proteins. Panel A: Fluorescence emitted by TNS binding (Ex/Em 322/465 nm) to native or guanidine hydrochloride (GuHCl) denatured DBP to measure the level of protein denaturation. Panel B: Native DBP or GuHCl DBP (both at 0.4 mg/ml) were allowed to form complexes with either 1.3 mg/ml C3 (physiological concentration), 0.26 mg/ml C3 or no C3 in the presence of factor B (fB, 0.2 mg/ml), factor D (fD, 1 μg/ml) and 0.5 mM Mg2+. All proteins were mixed at physiological (plasma) ratios with the exception of reduced or no C3 samples. The samples were incubated at 37°C for 15 minutes. Aliquots were analyzed using SDS-PAGE with β-mercaptoethanol on an 8% gel and then immunoblotted for DBP and C3 (for complex formation) and factor B (for complement activation). Panel C: Elastase inhibitory activity of α1PI was tested using the chromogenic substrate AAPV-pNA by measuring the release of pNA at 405 nm. Native and GuHCl denatured α1PI (both at 60 nM) were incubated at 37°C for 30 minutes with purified neutrophil elastase (60 nM), enzyme activity was then tested by adding 100 μM AAPV-pNA in assay buffer (0.1 M HEPES, pH 7.25 with 0.5 M NaCl, 0.05% Tween 20) and incubated at 37°C for another 30 minutes. Negative control is substrate alone without elastase, positive control is substrate and elastase only. AAPV-CMK sample is elastase pretreated with this peptide inhibitor as an additional control for inhibition of enzyme activity. Panel D: Native or GuHCl α1PI (both at 1 mg/ml) were allowed to form complexes with either 1.3 mg/ml C3, 0.26 mg/ml C3 or no C3 in the presence of factor B (fB, 0.2 mg/ml), factor D (fD, 1 μg/ml) and 0.5 mM Mg2+. All proteins were mixed at physiological (plasma) ratios with the exception of reduced or no C3 samples. The samples were incubated at 37°C for 15 minutes. Aliquots were analyzed using SDS-PAGE with β-mercaptoethanol on an 8% gel and then immunoblotted for α1PI and C3 and factor B. Panel E: Densitometry of the C3b:DBP complex bands in panel B (DBP blot) and C3b:α1PI complex bands in panel D (α1PI blot) comparing native versus GuHCl denatured proteins with the physiological (1.3 mg/ml) C3 concentration.

Article Snippet: Purified cobra venom factor (CVF), human serum depleted of individual complement components (C3, factor B and factor D), and purified human complement proteins (C3, factor B and factor D) were all purchased from Complement Technology, Inc. (Tyler, TX).

Techniques: Clinical Proteomics, Fluorescence, Binding Assay, Concentration Assay, Incubation, SDS Page, Activation Assay, Activity Assay, Purification, Negative Control, Positive Control, Control, Inhibition

Thermally denatured proteins activate the complement system via the alternative pathway. NHS was heated to 60°C for 1 hour (Δ serum) and then added to equal volume of either fresh NHS (lanes 2), factor B depleted serum (lanes 5), factor D depleted serum (lanes 6) or C3 depleted serum (lanes 7) and incubated for either 15 or 60 minutes at 37°C. The negative control was untreated NHS (lanes 1) or NHS that received equal amount of Δ serum in the presence of 10 mM EDTA (lanes 3) to prevent complement activation. The positive control was a mixture of Δ serum plus fresh serum treated with CVF (lanes 4) to activate the alternative pathway. Samples were separated using an 8% SDS-PAGE gel and blotted for factor B (alternative pathway activation and generation of the 33 kDa Ba fragment), C3 (110 kDa α-chain cleavage and 67 kDa iC3b generation) and α1PI (iC3b:α1PI complex formation).

Journal: Molecular immunology

Article Title: Enhanced recognition of plasma proteins in a non-native state by complement C3b. A possible clearance mechanism for damaged proteins in blood

doi: 10.1016/j.molimm.2014.10.022

Figure Lengend Snippet: Thermally denatured proteins activate the complement system via the alternative pathway. NHS was heated to 60°C for 1 hour (Δ serum) and then added to equal volume of either fresh NHS (lanes 2), factor B depleted serum (lanes 5), factor D depleted serum (lanes 6) or C3 depleted serum (lanes 7) and incubated for either 15 or 60 minutes at 37°C. The negative control was untreated NHS (lanes 1) or NHS that received equal amount of Δ serum in the presence of 10 mM EDTA (lanes 3) to prevent complement activation. The positive control was a mixture of Δ serum plus fresh serum treated with CVF (lanes 4) to activate the alternative pathway. Samples were separated using an 8% SDS-PAGE gel and blotted for factor B (alternative pathway activation and generation of the 33 kDa Ba fragment), C3 (110 kDa α-chain cleavage and 67 kDa iC3b generation) and α1PI (iC3b:α1PI complex formation).

Techniques: Incubation, Negative Control, Activation Assay, Positive Control, SDS Page

C3 dependent complement activators generate C5a in thermally denatured serum. Untreated NHS (Control) or NHS plus 60°C heated serum (Δ Serum) were treated with either CVF, zymosan, HAGG or buffer (Sham) for 60 minutes at 37°C and C5a levels were quantified by ELISA.

Journal: Molecular immunology

Article Title: Enhanced recognition of plasma proteins in a non-native state by complement C3b. A possible clearance mechanism for damaged proteins in blood

doi: 10.1016/j.molimm.2014.10.022

Figure Lengend Snippet: C3 dependent complement activators generate C5a in thermally denatured serum. Untreated NHS (Control) or NHS plus 60°C heated serum (Δ Serum) were treated with either CVF, zymosan, HAGG or buffer (Sham) for 60 minutes at 37°C and C5a levels were quantified by ELISA.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Pooled normal human serum was sham-treated with PBS (lane 1) or complement was activated by incubating serum at 37°C using either 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), 0.5 mg/300 μl heat-aggregated human IgG (lane 4). As a control to inhibit complement activation, 10 mM EDTA was added to serum prior to addition of CVF (lane 5). Serum aliquots were separated on an 8% SDS-PAGE and then immunoblotted for DBP, α1PI or α1AG. Arrows indicate the position of high molecular weight SDS-resistant bands formed during complement activation. The same samples also were blotted for factor B (fB) to verify cleavage as an indicator of complement activation (bottom panel).

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Pooled normal human serum was sham-treated with PBS (lane 1) or complement was activated by incubating serum at 37°C using either 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), 0.5 mg/300 μl heat-aggregated human IgG (lane 4). As a control to inhibit complement activation, 10 mM EDTA was added to serum prior to addition of CVF (lane 5). Serum aliquots were separated on an 8% SDS-PAGE and then immunoblotted for DBP, α1PI or α1AG. Arrows indicate the position of high molecular weight SDS-resistant bands formed during complement activation. The same samples also were blotted for factor B (fB) to verify cleavage as an indicator of complement activation (bottom panel).

Article Snippet: Human serum depleted of individual complement components (C3, C4, C5, factor B, factor D, properdin, factor H and factor I), and purified human complement proteins (C3, C3b, factor B, factor D, factor H and factor I) were all purchased from Complement Technology, Inc.

Techniques: Control, Activation Assay, SDS Page, High Molecular Weight

Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Panel A: Aliquots were separated on an 8% SDS-PAGE and then immunoblotted for the indicated proteins. Gels were cropped to focus only on the high molecular weight SDS-resistant bands. Samples also were blotted for factor B to verify cleavage as an indicator of complement activation. The 75 kDa β-chain of C3 was utilized as a loading control. Panel B: C5a ELISA of complement activation in serum. Panel C: C5a ELISA of complement activation in citrated plasma.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Panel A: Aliquots were separated on an 8% SDS-PAGE and then immunoblotted for the indicated proteins. Gels were cropped to focus only on the high molecular weight SDS-resistant bands. Samples also were blotted for factor B to verify cleavage as an indicator of complement activation. The 75 kDa β-chain of C3 was utilized as a loading control. Panel B: C5a ELISA of complement activation in serum. Panel C: C5a ELISA of complement activation in citrated plasma.

Techniques: Clinical Proteomics, Activation Assay, SDS Page, High Molecular Weight, Control, Enzyme-linked Immunosorbent Assay

Panel A: Serum samples depleted of individual complement components (fB, fD, fP, C3, C5 and C4) were activated with CVF (416 U/ml) for 15 minutes at 37°C. Samples where complex formation was abolished were reconstituted with physiological levels of missing purified protein (fB, fD or C3) followed by activation with CVF. Aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI and α1AG. Samples also were immunoblotted for C3 β-chain band as a loading control and factor B to show complement activation. Panel B: Factor B-depleted and C3-depleted sera were reconstituted with purified C3b (1.3 mg/ml) and incubated at 37°C for 1, 3.5 or 5 minutes, separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI and α1AG. Samples also were blotted for factor B to show complement activation in C3-depleted serum reconstituted with C3b. CVF activated normal human serum was included as a positive control.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: Serum samples depleted of individual complement components (fB, fD, fP, C3, C5 and C4) were activated with CVF (416 U/ml) for 15 minutes at 37°C. Samples where complex formation was abolished were reconstituted with physiological levels of missing purified protein (fB, fD or C3) followed by activation with CVF. Aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI and α1AG. Samples also were immunoblotted for C3 β-chain band as a loading control and factor B to show complement activation. Panel B: Factor B-depleted and C3-depleted sera were reconstituted with purified C3b (1.3 mg/ml) and incubated at 37°C for 1, 3.5 or 5 minutes, separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI and α1AG. Samples also were blotted for factor B to show complement activation in C3-depleted serum reconstituted with C3b. CVF activated normal human serum was included as a positive control.

Techniques: Purification, Activation Assay, SDS Page, Control, Incubation, Positive Control

Panel A: The amount of C5a generated in reconstituted C3-depleted serum samples measured by ELISA. Antibody coated sheep erythrocytes were treated with either C3-depleted serum alone or C3-depleted serum reconstituted with 1.3 mg/ml native C3, hydroxylamine treated C3 (C3-NH2OH) or C3b and incubated at 37°C for 1 hour. The supernatant was removed and C5a levels quantified by C5a ELISA. Panel B: C3-depleted serum alone or reconstituted with native C3 or hydroxylamine treated C3 (C3-NH2OH), both at 1.3 mg/ml, were activated with CVF (416 U/ml) plus 0.4 mM Mg2+ for 15 minutes at 37°C. CVF activated normal human serum was included as a positive control. Aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI, α1AG, factor B and C3. Panel C: Pooled normal human serum was sham-treated with PBS (lane 1) or complement was activated by incubating serum at 37°C using either 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), 0.5 mg/300 μl heat-aggregated human IgG (lane 4). As a control to inhibit complement activation, 10 mM EDTA was added to serum prior to addition of CVF (lane 5). Serum aliquots were separated on an 8% SDS-PAGE and then immunoblotted for C3. Panel D: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Aliquots were separated on an 8% SDS-PAGE and then immunoblotted C3.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: The amount of C5a generated in reconstituted C3-depleted serum samples measured by ELISA. Antibody coated sheep erythrocytes were treated with either C3-depleted serum alone or C3-depleted serum reconstituted with 1.3 mg/ml native C3, hydroxylamine treated C3 (C3-NH2OH) or C3b and incubated at 37°C for 1 hour. The supernatant was removed and C5a levels quantified by C5a ELISA. Panel B: C3-depleted serum alone or reconstituted with native C3 or hydroxylamine treated C3 (C3-NH2OH), both at 1.3 mg/ml, were activated with CVF (416 U/ml) plus 0.4 mM Mg2+ for 15 minutes at 37°C. CVF activated normal human serum was included as a positive control. Aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI, α1AG, factor B and C3. Panel C: Pooled normal human serum was sham-treated with PBS (lane 1) or complement was activated by incubating serum at 37°C using either 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), 0.5 mg/300 μl heat-aggregated human IgG (lane 4). As a control to inhibit complement activation, 10 mM EDTA was added to serum prior to addition of CVF (lane 5). Serum aliquots were separated on an 8% SDS-PAGE and then immunoblotted for C3. Panel D: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Aliquots were separated on an 8% SDS-PAGE and then immunoblotted C3.

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, SDS Page, Control, Activation Assay, Clinical Proteomics

Panel A: Purified C3, fD, DBP and 0.5 mM Mg2+ were mixed in the presence or absence of fB at physiological concentrations and incubated at 37°C for either 15 or 30 minutes. Factor I-depleted serum sample incubated at 37°C for 15 minutes in the presence of Mg2+ was included as a positive control. Mixtures were immunoblotted for DBP (for complex formation) and factor B (for complement activation). Panel B: Purified fB, fD, DBP and 0.5 mM Mg2+ were mixed with either native C3 or hydroxylamine treated C3 (C3-NH2OH), at physiological ratios were incubated at 37°C for 15 minutes. Factor I depleted serum sample incubated at 37°C for 15 minutes in the presence of Mg2+ was used as a positive control. Samples were immunoblotted for DBP to assess complex formation and factor B to verify complement activation.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: Purified C3, fD, DBP and 0.5 mM Mg2+ were mixed in the presence or absence of fB at physiological concentrations and incubated at 37°C for either 15 or 30 minutes. Factor I-depleted serum sample incubated at 37°C for 15 minutes in the presence of Mg2+ was included as a positive control. Mixtures were immunoblotted for DBP (for complex formation) and factor B (for complement activation). Panel B: Purified fB, fD, DBP and 0.5 mM Mg2+ were mixed with either native C3 or hydroxylamine treated C3 (C3-NH2OH), at physiological ratios were incubated at 37°C for 15 minutes. Factor I depleted serum sample incubated at 37°C for 15 minutes in the presence of Mg2+ was used as a positive control. Samples were immunoblotted for DBP to assess complex formation and factor B to verify complement activation.

Techniques: Purification, Incubation, Positive Control, Activation Assay

Panel A: Purified C3, fB, fD and DBP were mixed with either factor H alone, factor I alone or a combination of factors H and factor I at physiological ratios and then activated with CVF in the presence of 0.5 mM Mg2+ for the indicated times. Samples were immunoblotted for DBP to assess complex formation and factor B to verify complement activation. C3 β-chain was used as a loading control. Panel B: The uncropped DBP immunoblot of the last six lanes of panel A to show intermediate cleavage products. Panel C: Purified C3, fB, fD and α1AG were mixed with either factor I alone or a combination of factors H and factor I at physiological ratios and then activated with CVF in the presence of 0.5 mM Mg2+ for the indicated times. Samples were immunoblotted for α1AG (center panel) or C3d (right panel) to assess complex formation and breakdown. The positions of C3b, iC3b and C3dg complexes with α1AG are indicated. To compare complex degradation in serum to the purified protein system, factor I depleted serum alone, or factor I depleted serum reconstituted with factor I and activated with CVF for 15 minutes, were separated and immunoblotted for α1AG (left panel).

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: Purified C3, fB, fD and DBP were mixed with either factor H alone, factor I alone or a combination of factors H and factor I at physiological ratios and then activated with CVF in the presence of 0.5 mM Mg2+ for the indicated times. Samples were immunoblotted for DBP to assess complex formation and factor B to verify complement activation. C3 β-chain was used as a loading control. Panel B: The uncropped DBP immunoblot of the last six lanes of panel A to show intermediate cleavage products. Panel C: Purified C3, fB, fD and α1AG were mixed with either factor I alone or a combination of factors H and factor I at physiological ratios and then activated with CVF in the presence of 0.5 mM Mg2+ for the indicated times. Samples were immunoblotted for α1AG (center panel) or C3d (right panel) to assess complex formation and breakdown. The positions of C3b, iC3b and C3dg complexes with α1AG are indicated. To compare complex degradation in serum to the purified protein system, factor I depleted serum alone, or factor I depleted serum reconstituted with factor I and activated with CVF for 15 minutes, were separated and immunoblotted for α1AG (left panel).

Techniques: Purification, Activation Assay, Control, Western Blot

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