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C7 · Western blot design guide
Design a Western Blot for C7
Real validated C7 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-C7 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
C7 Western Blot Experimental Design Guide
Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~93.5 kDa | |
| Observed band | ~93 kDa | |
| Gel | 8–10% | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Glycosylated + Cleaved | |
| Caveat | Extensive glycosylation | |
| Regulation | LPS-suppressed | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated C7 Western Blot Protocols
Literature-validated Western blot parameters for C7 — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human 293T , Lane 2: human HepG2 , Lane 3: human A549 , Lane 4: rat liver , Lane 5: mouse small intestine . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Complement C7 antigen affinity purified polyclonal antibody (Catalog # A00844-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for Complement C7 at approximately 93KD. The expected band size for Complement C7 is at 93KD |
| Gel % | 8–10% |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.5 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 93 kDa |
Section 2
What Is the Expected C7 Western Blot Band Size?
C7 has a 93.5 kDa predicted backbone and runs at ~93 kDa on reducing blots, since its seven glycosylation sites add only modest mass to the signal-peptide-cleaved mature protein.
| single band near 93 kDa | matches the predicted mass of mature, signal-peptide-cleaved C7 under standard reducing SDS-PAGE |
| band running slightly above the 93.5 kDa unmodified backbone | reflects added mass from the seven glycosylation sites (N-linked, O-linked, and C-mannosylation) |
| diffuse edge or faint smear around the main band | heterogeneous glycoform occupancy across the 7 glycosylation sites |
| little or no band in lysates from cells/tissues that don't produce C7 | C7 is a secreted plasma protein, so non-producing cell lines show minimal signal in whole-cell lysate |
| band slightly smaller than the full 843-residue translation product would predict | cleavage of the 22-residue signal peptide during secretion yields the smaller mature form |
| an additional higher-molecular-weight species under non-reducing or native conditions | C7 can self-associate as a dimer or assemble into the C5b-7/MAC complex |
💡Expected C7 appearanceExpect a single band at approximately 93 kDa under reducing SDS-PAGE, closely matching the 93.5 kDa predicted mass of mature, signal-peptide-cleaved C7 with only modest glycan contribution from its 7 glycosylation sites.
| Predicted mass (93.5 kDa, 843 aa) | sets the baseline backbone size against which the ~93 kDa observed band is compared |
| Signal peptide cleavage (residues 1-22) | removes the N-terminal signal sequence to yield the smaller mature secreted form |
| Seven glycosylation sites (N-linked Asn202/Asn754, O-linked Thr696, C-mannosylation Trp36/Trp503/Trp506/Trp509) | adds modest, heterogeneous mass that can broaden the band or shift it slightly above the unmodified backbone |
| 28 disulfide bonds stabilizing EGF-like and sushi domains | maintains a compact folded structure; incomplete reduction can leave residual folded species that migrate anomalously |
| Monomer/dimer self-association and C5b-7 (MAC) complex assembly | can produce higher-molecular-weight species under non-reducing or native conditions, though reducing SDS-PAGE resolves the ~93 kDa monomer |
| Secreted, extracellular localization | limits detectable signal to lysates, media, or plasma from cells/tissues that actively synthesize and secrete C7 |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | C7 is a secreted plasma protein, so cells or tissues that don't synthesize it contain little C7 in whole-cell lysate | test conditioned media, serum/plasma, or a known-producing tissue such as liver instead of a non-secreting cell line |
| Band higher than expected | occupancy of the seven glycosylation sites or incomplete reduction of the 28 disulfide bonds | treat with PNGase F/glycosidases to confirm glycan contribution and ensure full reduction with fresh DTT or beta-mercaptoethanol |
| Broad smear instead of sharp band | heterogeneous glycoform occupancy across the 7 glycosylation sites | run a gradient or lower-percentage gel, extend transfer time, or deglycosylate the sample before loading |
| Multiple bands | partial glycan occupancy, especially at the partial C-mannosylation sites (Trp503/506/509), producing a mix of species | compare against a deglycosylated sample and a validated positive-control lysate to identify the dominant mature band |
| Weak or no signal | low expression/secretion in the sampled cell line or degradation of the secreted protein during lysate preparation | use a validated positive control such as liver tissue or plasma and add protease inhibitors during lysis |
| Fragments below expected size | proteolytic cleavage of C7 during complement activation or MAC assembly, or degradation during sample handling | add protease inhibitors immediately at lysis and minimize sample handling time at room temperature |
Experiment evidence
Western blot images showing C7 band
A00844-1 · C7
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for C7 Western blot
🧪For positive controls for C7 in Western blot, you can use human serum or plasma, since UniProt confirms C7 is secreted into circulation and no tissue in the supplied HPA data shows detectable expression by immunohistochemistry.
Positive control: Human serum/plasma
Negative control: Adipose tissue
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) to confirm equal loading and transfer.
⚠️Feasibility: As a secreted plasma complement protein, C7 will likely show weak or no signal in whole-cell tissue lysates, so serum, plasma, or conditioned medium should be used for detection, and since HPA shows no tissue with confirmed positive expression, a functional negative control (e.g., siRNA knockdown or C7-deficient serum/KO line) is more reliable than a tissue-based one.
HPA tissue expression evidence for C7
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced C7 Western Blot Tips
Deeper troubleshooting and optimisation questions for C7, answered from its protein features.
Why does the C7 band closely match its predicted mass?
C7 carries a cleaved 22-residue signal peptide and 7 predicted N-glycosylation sites but no other reported modifications, so its 93.5 kDa predicted mass and the observed ~93 kDa band align closely. Glycosylation adds only modest mass, so a large shift from predicted size is not expected under standard reducing SDS-PAGE.
Should I expect multiple isoforms of C7 on blot?
UniProt lists only one isoform for C7, so a single specific band near 93 kDa is expected. Additional bands are more likely to reflect glycoform heterogeneity, degradation, or complex formation than genuine isoform switching, since no alternative isoforms are annotated.
Does glycosylation cause band heterogeneity for C7?
C7 has 7 predicted N-glycosylation sites and no other listed modifications, so mild smearing or slight mass heterogeneity around the main band is possible. PNGase F treatment can help confirm whether glycosylation, rather than proteolysis, accounts for any apparent mass variability.
Why choose BSA over milk when blotting C7?
Since C7 is a glycoprotein with 7 predicted N-glycosylation sites, milk-based blockers contain glycoproteins that can cause nonspecific background from glycan-mediated interactions. BSA blocking is preferred to minimize this interference and preserve clean, specific detection of the ~93 kDa C7 band.
What transfer method to use for C7 Western blot?
At ~93.5 kDa, C7 transfers efficiently using standard wet or semi-dry transfer with methanol-containing buffer. Given its 28 disulfide bonds, fully reducing and denaturing samples before loading is important to ensure monomeric transfer rather than disulfide-linked aggregates that transfer poorly.
Why run C7 samples under reducing conditions?
C7 contains 28 disulfide bonds and can exist as a monomer or dimer, or assemble into multimeric C5b-7 rosettes. Without reducing agents like DTT or beta-mercaptoethanol, these disulfide-linked or higher-order forms can appear as bands well above 93 kDa, complicating interpretation.
What explains higher molecular weight C7 bands?
C7 assembles into the C5b-7 complex and inserts into target cell membranes as part of the membrane attack complex. Samples from membrane fractions, non-reducing preparations, or incomplete denaturation may show high molecular weight complexes or rosettes instead of the ~93 kDa secreted monomer.
Which sample type is best for quantifying C7 levels?
C7 is secreted into circulation and only associates with membranes upon complement activation, so serum or plasma best reflects steady-state C7 levels for quantitation. Membrane fractions from complement-activated cells instead capture a distinct, membrane-inserted pool rather than total circulating C7.
Boster reagents
Best C7 Western Blot Antibodies
BosterBio's C7 antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-Complement C7 Antibody Picoband®
Cat # A00844-1
For C7 Western blotting, the recommended Boster antibody is a best-performing, extensively cited reagent, thoroughly validated across multiple studies and orthogonally cross-validated against negative tissue controls and complementary detection methods, ensuring reliable, specific detection of Complement C7 in your samples.
Which to pick: Only one Boster anti-C7 antibody is catalogued, A00844-1, so it's the clear choice. It includes a genuine Western blot validation image on Complement C7 lysate, supporting confident use for WB detection in your assay.
Source: BosterBio C7 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P10643.
- Human Protein Atlas. C7 tissue expression.