C6 Protein, CAS No.80295-56-3

CAS: 80295-56-3 Cat. No.: C501107 Peso molecular: 105,000 Da (single chain)

GRADE & PURITY Moligand™, PBS Only, 1.0 mg/mL, 0.22 µm filtered

Bioactivity

>70% versus normal human serum standard

Visão geral

Product description：
Purity: >90% by SDS-PAGE

Extinction Coeff. A280 nm = 1.08 at 1.0 mg/mL

Molecular Weight: 105,000 Da (single chain)

Precautions: Use normal precautions for handling human blood products

General Description

Native human C6 is a naturally glycosylated (11%) protein composed of a single polypeptide chain of 105,000 Da. C6 is essential for formation of the membrane attack complex (MAC) and is activated non-proteolytically by binding to recently-formed C5b at the cell membrane. Each pathway of complement activation generates proteolytic 

enzyme complexes (C3/C5 convertases) which are bound to the target surface (Law, S.K.A. and Reid, K.B.M. (1995); Ross, G.D. (1986)). These enzymes cleave a peptide bond in the larger alpha chain of C5 releasing the anaphylatoxin C5a and activating C5b. This is the only proteolytic step in the assembly of the C5b-9 complex. C5b is unstable, but it remains bound to the activating complex for a brief time (~2 min) during which it either binds a single C6 from the surrounding fluid or it decays and is no longer capable of forming MAC. The C5b,6 complex may also remain bound to the C3/C5 convertase where the binding of a single C7 exposes a membrane-binding region and C5b,6,7 can 

partially insert into the bilipid layer of the target cell. Up to this point the complex may diffuse away from the target cell and enter the membrane of a nearby cell. This is called bystander lysis or “reactive lysis” and can be a significant source of pathology. Each C5b-7 complex can bind one C8 protein molecule which results in the complex inserting more firmly into the membrane. This complex binds C9 and each bound C9 can bind another C9 initiating formation of a ring structure containing up to 18 C9 molecules (Podack, E.R. (1984)). C5b-9 complexes with one or more C9 are referred to as the Membrane Attack Complex (MAC) of complement. Not all C5b-8 complexes have 

complete rings of C9 with the average being only three C9 per C5b-8 complex. Completed protein rings of C9 form the pores seen on electron micrographs and they result in leakage of metabolites and small proteins out of the cell as well as movement of water into the cell. If sufficient numbers are inserted into a cell membrane then water flowing into the cell, due to osmotic pressure, will rupture the cell membrane allowing the entire contents of the target cell (or a bystander cell) to be released. Either process may result in cell death. Originally it was thought that this required only one C5b-9 complex per cell (referred to as the “one hit theory” of lysis (Rommel F.A. and Mayer, M.M. (1973)), but this is probably not correct. For example, an erythrocyte requires ~850 C5b-9 complexes, as measured by the number of C7 molecules, for lysis to occur (Bauer, J. et al. (1979)). Host cells protected from MAC by CD59 require sufficient numbers of C5b-9 to tie up all the CD59 and then ~850 C5b-9 in addition. Lysis of nucleated cells requires many more C5b-9 complexes due to their size and due to the presence of multiple defense mechanisms in such cells.

Physical Characteristics & Structure
Molecular weight: 105,000 Da composed of a single polypeptide chain. The pI of C6 is heterogeneous from 6.0 to 6.5.

Function

See General Description above.

Assays

The simplest assay for C6 is to use C6-depleted human serum and measure the lysis of EA (classical pathway) or Er (alternative pathway) as a function of the concentration of added test sample or standard purified C6. Each unique application might require appropriate conditions to be determined. However, a typical assay would involve mixing on wet ice 25 µL C6-Dpl, C6-containing sample diluted with GVB++ to contain from 0.1 to 1 ng C6 , and sufficient GVB++ to bring the volume to 300 µL. EA (3 X 107 cells in 200 µL) diluted in GVB++ should be added last. Purified C6 or normal human serum (NHS) may be used as a source of C6. The reaction mixture is incubated for 30 min at 37℃ and 1 mL of cold GVBE added, mixed and centrifuged to spin out unlysed cells. The released hemoglobin in the supernatant is then analyzed at 415 nm and compared to blanks without C6 (background lysis control) and cells incubated with 275 µL water instead of GVB++ and 25 µL C6-Dpl (100% lysis control).

Many other assays have been described using EA preloaded with C1 (EAC1 cells) or preloaded with the classical pathway C5 convertase (EAC1423 cells), however, all these assays require the use of multiple purified complement components or more difficult-to-prepare reagents (Dodds, A.W. and Sim, R.B. (1997); Morgan, B.P. (2000); Tack, B.F., et al. (1981)).

Applications

See General Description above.

In vivo
The normal serum concentration of C6 is 64 µg/mL (normal range 54 to 72µg/mL). The primary site of synthesis is the liver. C6 is an acute phase protein and its synthesis is stimulated by the cytokines that stimulate increased biosynthesis of many other complement proteins.

Regulation

Many proteins and other components of plasma have an inhibitory effect on the 

lytic activity of C5b-9 complexes but there are no specific C6 inactivators. Most of the C5b-9 inhibitors interact with the complex after the C5b-7 stage. If any of the C5b-containing complexes fail to insert into a membrane they may self-aggregate or bind to regulatory proteins the most prevalent of which is S Protein. S protein (also called 

vitronectin) is an 80,000 Da plasma protein that binds to C5b-9 complexes that fail to insert in the target cell membrane. This reduces damage to nearby host cells. Many other serum components inhibit or partially inhibit lysis by C5b-9 and these include SP40,40 (also known as clusterin and apolipoprotein J) and many plasma lipoprotein complexes (LDL, HDL, etc.).

Host cells protect themselves from C5b-9 by a variety of mechanisms. Membrane proteins DAF, MCP, and CR1 inhibit formation of C3/C5 convertases preventing MAC formation. CD59, also called “homologous restriction factor” and “protectin”, is an18,000 to 20,000 Da ubiquitous component of cell membranes that is very effective at binding to and inhibiting the lytic potential of C5b-8 and C5b-9 complexes. The species-specificity of CD59 is not absolute and many mammalian CD59 proteins do inhibit or partially inhibit MAC from other species. The specificity that is observed appears to be due to incompatibilities between C8 of one animal and the CD59 of another. Like DAF,CD59 contains a GPI anchor (a post-translationally added lipid tail that inserts into the bilipid layer of the cell). The disease PNH is caused by the loss of enzymes that attach the GPI tail, thus depriving cells of the ability to inactivate C3/C5 convertases and the ability to inactivate C5b-9. This results in complement-mediated damage to and eventual lysis of long-lived blood cells such as erythrocytes and platelets.

Genetics

Human chromosome location 5q 12-14. Accession number HSC6A. Mouse chromosome 15. Human genomic structure: the gene spans 80 kb with 18 exons.

Deficiencies
Human deficiencies have been found and exhibit autosomal recessive transmission. Patients generally exhibit abnormally high susceptibility to recurrent meningococcal meningitis and systemic neisserial infections. Partial deficiencies do not seem to show adverse clinical effects.

Diseases
See Deficiencies above.

Precautions/Toxicity/Hazards
This protein is purified from human plasma, therefore precautions appropriate for handling any blood-derived product must be used even though the source was shown by certified tests to be negative for HBsAg, HTLV-I/II, STS, and for antibodies to HCV, HIV-1 and HIV-II.

Hazard Code: B    WGK Germany 3

MSDS available upon request.

Specifications

Product Name

C6 Protein, CAS No.80295-56-3

Grau

Moligand™, PBS Only

Especificações e pureza

Moligand™,PBS Only,1.0 mg/mL, 0.22 µm filtered

Bioatividade

>70% versus normal human serum standard

Fonte

Normal human serum (shown by certified tests to be negative for HBsAg and for antibodies to HCV, HIV-1 and HIV-II)

CAS

80295-56-3

Tipo de molécula

Protein

Armazenamento e envio

Forma

Liquid

Concentração

1.0 mg/mL, 0.22 µm filtered

Condições de armazenamento de armazenamento

Store at -80°C,Avoid repeated freezing and thawing

Enviado em

Dry ice packs + Cold packs

Application Protocols

Tested applications, recommended dilutions, and positive-control materials are not specified for this item; refer to the CoA/Spec Sheet.

General note: Because this section requires item-specific validation, Aladdin provides protocol guidance on the product documentation when available.

Biological Roles

C6 is a key component of the terminal complement pathway, functioning in immune defense by enabling assembly of the membrane attack complex (MAC) (literature/general).

• Pathway context: After activation of the classical, lectin, or alternative pathways, C5 convertase generates C5b, which binds C6 to form the stable C5b6 complex. This complex recruits C7, C8, and polymerizing C9 to form MAC (C5b–9) that can perforate target membranes (literature).
• Molecular function: C6 provides structural modules necessary for high-affinity binding to C5b and for orchestrating conformational transitions that enable subsequent component recruitment (literature).
• Structural biology: Electron microscopy and crystallographic studies place C6 within the MACPF/CDC-related superfamily, sharing a fold capable of large conformational rearrangements during pore formation (literature).
• Regulation and checks: Host regulators (e.g., CD59) act downstream to prevent off-target lysis; factor H/I regulate earlier steps. Studying purified C6 helps dissect these regulatory nodes by selective reconstitution (literature).
• Systems biology uses: Quantifying C6 levels and function contributes to complement profiling in serum or plasma and supports mechanistic studies of innate immunity (literature).

Notes for this item:

• This listing provides C6 Protein as a research reagent in PBS (per Product Data). No activity, purity, or source species details are specified here—consult the CoA/Spec Sheet for lot-specific information.

Buffer Applications

This item is supplied in PBS only (per Product Data). Practical buffer considerations for experimental use (general/literature):

• Complement functional assays:
  • For classical-pathway hemolysis, exchange into barbitone/Veronal buffer containing Mg2+ and Ca2+ (often termed GVBS++) to support optimal complement activity. Maintain ionic strength carefully; chelators (EDTA/EGTA) inhibit complement.
  • For alternative-pathway settings, Mg2+ is essential while Ca2+ may be omitted (EGTA-Mg2+ conditions) depending on the assay design.
• Biophysical assays:
  • PBS is usually acceptable for ELISA, WB sample prep, or BLI/SPR running buffers if compatible with the sensor chemistry. Filter (0.22 µm) and degas to reduce baseline noise.
  • For label conjugation, avoid primary-amine buffers (e.g., Tris) when using NHS esters; PBS or bicarbonate is typically suitable.
• Stability additives (as needed):
  • Trace nonionic detergents (e.g., 0.01–0.05% Tween-20) can reduce nonspecific adsorption in immunoassays; assess impact on functional complement assays before use.
  • Carrier proteins are sometimes added to minimize loss at low ng–µg/mL levels; none are present in this item per “PBS Only.” Add externally only if compatible with your readout.

Implementation tips:

• If buffer exchange is required, use gentle methods (spin columns, dialysis, SEC) at 2–8°C to preserve activity.
• Validate pH (target near neutral unless your assay prescribes otherwise) and ensure sterility for cell-associated studies.

Green Alternatives

For a protein reagent in PBS, “green” considerations center on formulation, handling, and cold-chain logistics rather than solvent replacement.

• Current profile: Aqueous PBS formulation (no organic solvents stated). This is inherently low-VOC and aligns with greener lab practices.
• Opportunities to reduce footprint (general):
  • Optimize aliquot sizes to minimize freeze–thaw and reduce wastage.
  • Consolidate shipments to limit dry-ice usage; ensure recipient freezers are prepared to avoid re-shipments.
  • Employ energy-efficient ultra-low freezers and temperature mapping to maintain -80°C with minimal power.
  • Use recyclable secondary packaging and avoid overuse of absorbents when permissible by regulations.
• Assay design: Prefer microplate-scale assays and microscale biophysics (SPR/BLI) to reduce protein consumption.

Trade-offs:

• Additional stabilizers (e.g., carriers, preservatives) can extend bench stability but may complicate downstream assays or introduce bioburden concerns; this item is “PBS Only,” avoiding such additives.
• Dialysis/buffer exchange to specialized assay buffers introduces consumable waste; balance data needs with environmental impact.

Summary: The product already embodies low solvent impact; sustainability gains come from right-sizing, careful cold-chain management, and assay miniaturization.

Pharmaceutical Uses

This product is designated for research use only (per Product Data) and is not intended for human or veterinary use.

In a pharmaceutical R&D and analytics context (general, non-clinical):

• Analytical standards/controls: Purified C6 can serve as a system suitability or positive control material in immunoassays (e.g., ligand binding assays) and LC–MS proteomics methods that quantify complement components.
• Process development studies: Complement proteins are sometimes used to stress-test biomaterial surfaces or evaluate complement-activation potential of formulations in vitro. Purified C6 contributes to defined reconstitution panels.
• Mechanistic research: Supports nonclinical investigations of innate-immune interactions with biologics, delivery vehicles, or implant coatings under controlled in vitro conditions.

No pharmacopeial monograph or excipient role is specified for this item. Any use beyond basic research should be risk-assessed and supported by internal quality documentation (CoA, method suitability).

Physical Properties

Item-specific physicochemical specifications are not provided for this product listing; consult the CoA/Spec Sheet for lot-resolved details.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Concentration/formulation: Grade indicates “PBS Only”; precise concentration, pH, and excipients are not specified here.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
  • Literature: human C6 migrates ~100–110 kDa by SDS-PAGE due to glycosylation heterogeneity.
• Solubility: Supplied in phosphate-buffered saline (PBS) per Product Data; ready for use in aqueous systems. Avoid organic solvents that denature proteins.
• pH: Not specified for this item.
  • Literature: PBS typically ~pH 7.2–7.4 (buffer-system property).
• Extinction coefficient, isoelectric point, glycan content: Not specified for this item; refer to CoA/Spec Sheet.
• Thermal behavior: As with most complement proteins, C6 is heat-labile; maintain on ice during handling (general protein guidance). No denaturation temperature is specified for this item.

Interpretation guidance (general):

• Proteins do not have meaningful BP/MP; stability is governed by tertiary/quaternary structure.
• Viscosity and refractive index are dominated by the aqueous PBS vehicle; no unique item-specific values are provided here.

Quality and Grades

• Stated grade/purity: Moligand™, PBS Only (per Product Data).
  • Moligand™: Proprietary Aladdin designation. Specific acceptance criteria (purity by SDS-PAGE, endotoxin, host-cell protein/DNA, activity) are not listed here—refer to the CoA/Spec Sheet for measured values and test methods.
  • PBS Only: Formulated in phosphate-buffered saline without additional carriers or stabilizers stated. This is advantageous where carrier proteins (e.g., BSA) or preservatives (e.g., sodium azide) would interfere with functional or biochemical assays. Exact buffer composition (pH, ionic strength) is not specified here.

What this means in practice:

• Expect a protein reagent intended for research workflows requiring clean background and predictable buffer composition.
• For quantitative assays (e.g., ELISA standard curves, hemolytic complement assays), verify lot-specific concentration, purity, and any residual contaminants on the CoA.
• If sterility/endotoxin are critical (e.g., cell-based assays), confirm these attributes; they are not specified for this item.

Documentation and traceability:

• Batch-specific CoA should define identity (e.g., immunoreactivity), purity (densitometry), and storage conditions. Any stabilizers or excipients, if present, will be disclosed on the CoA.
• For regulatory or method-validation contexts, align your acceptance criteria (purity, activity windows) with the CoA values for your lot.

Reaction and Applications

As a purified complement protein, C6 is primarily used in biochemical and immunological assays rather than in synthetic organic “reactions.” Typical research applications (literature/general) include:

• MAC assembly studies: Combine with C5b (as C5b6 complex) and downstream components (C7, C8, C9) to reconstitute the membrane attack complex in vitro. Useful for kinetic analyses, stoichiometry determination, and structure–function work.
• Hemolytic assays: Reconstitute activity in C6-deficient serum to measure classical (CH50) or alternative (AH50) pathway lysis of sensitized erythrocytes. C6 concentration–response curves can quantify functional potency.
• Binding/interaction assays: Study C6 engagement with C5b and subsequent partners using SPR, BLI, MST, or co-immunoprecipitation.
• Immunoassay standards/controls: Use as a calibrator or positive control for anti-C6 ELISA, western blot, or mass spectrometry method development (verify compatibility with your assay matrix).
• Inhibition/neutralization tests: Evaluate inhibitors or antibodies targeting the terminal complement pathway by monitoring MAC formation in presence/absence of C6.

Practical tips (general):

• Maintain on ice; pre-chill plasticware. Use low-binding consumables to minimize adsorption.
• Calibrate protein concentration by UV absorbance only if the extinction coefficient for your lot is provided on the CoA; otherwise use amino-acid analysis or BCA with appropriate standards.
• For reconstitution experiments, ensure other complement components are present at nonlimiting levels and that buffer contains requisite divalent cations for pathway-specific assays.

Reaction Conditions

Classical chemical reaction conditions are not applicable. For functional reconstitution experiments with complement components (literature/general):

• MAC reconstitution: Assemble C5b6 with downstream C7, C8, and C9 at near-neutral pH, physiological ionic strength, and low temperatures (on ice) for staging; incubation at 25–37°C often promotes complex formation and activity. Include Mg2+ and Ca2+ as dictated by pathway-specific requirements.
• Hemolytic assays: Typical incubation 30–60 min at 37°C with sensitized erythrocytes; readouts via hemoglobin release at 541 nm. Optimize C6 stoichiometry to be rate-limiting or saturating depending on the experimental aim.
• Binding studies (SPR/BLI): Run in filtered PBS or HEPES-buffered saline with minimal surfactant; temperature 25°C is common. Regeneration conditions must be validated empirically to avoid denaturation.

These are general literature guidelines. Exact conditions, concentrations, and expected activities for this item are not specified; consult your method SOPs and verify with small-scale pilots.

Safety and Handling

• GHS classification / pictograms / H-statements: Not specified for this item; refer to the SDS for authoritative hazard information.
• Expected hazard profile (general for purified proteins in PBS): Typically low acute toxicity; treat as a laboratory chemical. Avoid ingestion, inhalation of aerosols, and contact with skin/eyes. Proteins may elicit allergic responses in sensitized individuals.
• PPE: Lab coat, disposable nitrile gloves, safety glasses. Work in a biosafety cabinet if aerosol generation is possible or if sterility must be maintained.
• Engineering controls: Keep containers closed. Use on ice to limit activity loss. Utilize secondary containment during transport between freezers and bench.
• Handling:
  • Thaw on ice. Mix gently by end-over-end inversion; avoid vigorous vortexing or frothing.
  • Use low-protein-binding tubes and tips to minimize adsorption losses.
  • Avoid repeated freeze–thaw cycles (per Product Data); aliquot upon first thaw.
• Incompatibilities (general): Strong oxidants, proteases, extreme pH (<5 or >9), high ionic detergents, and organic solvents can denature proteins. Sodium azide and other preservatives may interfere with downstream functional assays; none are specified for this item.
• First aid (overview): Skin/eye contact—rinse with water. Inhalation—move to fresh air. Ingestion—rinse mouth. Seek medical attention as needed. Defer to the SDS for full instructions.
• Waste: Dispose according to institutional guidelines for nonhazardous aqueous protein solutions unless local rules classify otherwise.

Solvent Selection

For this protein reagent, “solvent” refers to the aqueous buffer system. This item is supplied in PBS only (per Product Data), which suits most biochemical workflows.

• Polarity / miscibility: Aqueous, fully miscible with water-based buffers of compatible pH and ionic strength.
• Why PBS?
  • Maintains near-physiologic pH and ionic strength, supporting protein stability and complement protein integrity (general).
  • Compatible with many immunoassays and biochemical protocols.
• When to consider alternative buffers (general guidance):
  • Functional complement assays often include divalent cations; use barbitone/Veronal buffer containing Mg2+ and Ca2+ for classical pathway hemolysis tests (literature). If needed, dialyze/exchange from PBS into assay buffer.
  • For conjugation chemistry (e.g., amine-reactive labeling), switch to amine-free buffers (e.g., phosphate or bicarbonate without Tris or primary amines) as appropriate.
  • For storage stability, small amounts of inert carrier (e.g., 0.1% HSA) can reduce adsorption losses; none are present in this item per “PBS Only”. Add only if compatible with your application.

Small comparison (general):

• PBS: broad compatibility; may chelate-sensitive assays minimally affected; lacks divalent cations by default.
• Veronal (with Mg2+/Ca2+): favored for hemolytic complement assays; not ideal for some cell culture contexts.
• HEPES: superior buffering near pH 7.4 for live-cell work; verify compatibility with your readout.

Storage and Reconstitution

• Storage conditions (per Product Data): Store at -80°C. Avoid repeated freezing and thawing. Shipments are made on dry ice packs + cold packs to maintain the cold chain.
• Aliquoting: Upon first thaw, divide into single-use aliquots in low-protein-binding tubes to prevent activity loss from freeze–thaw.
• Thawing/handling: Thaw on ice. Mix gently by inversion; avoid vigorous vortexing and bubbles.
• Short-term stability: If bench work is required, keep on ice and return promptly to -80°C after use. Refrigerated storage (2–8°C) should be minimized and validated empirically; no time limits are specified for this item.
• Reconstitution: Not specified for this item; refer to CoA/Spec Sheet. If received lyophilized (appearance not specified), a typical approach is gentle reconstitution with cold PBS to the desired concentration, followed by clarification spin at 4°C. Validate function post-reconstitution.
• Sterility: Not specified. If sterility is required, work aseptically and, if compatible, sterile-filter through 0.22 µm low-protein-binding membranes.
• Adsorption mitigation: For very low use concentrations, pre-coat plasticware or add a compatible carrier if your assay allows (none included in this item per “PBS Only”).

Always consult the lot-specific CoA/Spec Sheet for definitive instructions on concentration, buffer composition, and any additional stabilizers.

Structure and Identity

C6 Protein is a large human complement protein (terminal pathway component) supplied as a research reagent.

• CAS: 80295-56-3 (protein entry, literature identifier)
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
  • Typical literature value: single-chain glycoprotein ~100–110 kDa depending on glycosylation (literature).
• SMILES / InChI / InChIKey: Not applicable to proteins of undefined sequence/heterogeneity in catalog context.
• Protein family: Complement component of the membrane attack complex (MAC) pathway (literature).
• Structural features (literature):
  • Modular, multi-domain glycoprotein; contains MACPF/CDC-like domain architecture enabling assembly with C5b and downstream components (literature).
  • N-linked glycosylation; domain interfaces that engage C5b to form C5b6 and nucleate MAC assembly (literature).
• 2D description in words: A single polypeptide chain folded into several globular domains connected by flexible linkers; external carbohydrate moieties; surface patches mediating protein–protein interactions with C5b and subsequent complement components (literature).

Notes on identity vs. this item:

• Grade/Purity: Moligand™, PBS Only (per Product Data). No additional stabilizers, carriers, or tags are stated; confirm on CoA.
• This listing denotes a protein reagent; small-molecule identifiers (e.g., SMILES) are not meaningful here.

Synthetic Utility

Not typically applicable. C6 Protein is a biological macromolecule used for biochemical and immunological research, not as a reagent or building block in synthetic organic chemistry.

For users exploring chemical biology (general):

• Conjugation chemistries (e.g., NHS–ester labeling of lysines, maleimide–thiol coupling to engineered cysteines) may be applied to C6 for probe development, provided activity is verified post-labeling.
• Crosslinkers (e.g., BS3, DSS) can map interaction interfaces within C5b–C6 assemblies, followed by MS analysis. These are specialized workflows outside routine “synthetic utility.”

Target Specificity

Not applicable to this listing. This product is C6 Protein itself and not an antibody or affinity reagent. No clone, isotype, antigen, or species-reactivity data are provided in the Product Data.