C5 Antibody (Mouse mAb) [J8C4]

Catalog No.: F3817

Application: Reactivity: Human

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代表番号: 045-509-1970|電子メール：sales@selleck.co.jp

使用情報

Dilution

Application
IHC, IF, FCM, ELISA

Source
Mouse Monoclonal Antibody

Reactivity
Human

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
188 kDa

プロトコール

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

生物学的記述

Specificity
C5 Antibody (Mouse mAb) [J8C4] detects endogenous levels of total C5 protein.

Uniprot ID
P01031

Clone
J8C4

Synonym(s)
C5; complement component 5; complement C5; CPAMD4; anaphylatoxin C5a analog; C3 and PZP-like alpha-2-macroglobulin domain-containing protein 4; FLJ17816; FLJ17822; MGC142298;

Background
Complement component C5 is a central effector protein of the complement cascade that links upstream C3 convertase activity to generation of potent anaphylatoxin and cytolytic responses, and belongs to the family of large modular complement proteins that circulate mainly as inactive precursors until they are cleaved by C5 convertases on activating surfaces. The molecule consists of two chains derived from a single precursor and organized into multiple β‑sheet‑rich domains homologous to those in C3 and other terminal complement components, creating surfaces that support recognition by C5 convertases and that, after cleavage, yield a soluble effector fragment and a membrane‑binding fragment that initiates terminal complex assembly. Conversion of C3 into C3b during classical, lectin, or alternative pathway activation allows assembly of pathway‑specific C5 convertases, which bind native C5 and cleave it into the small fragment C5a and the larger fragment C5b; C5a diffuses away and acts as a strong anaphylatoxin, whereas C5b remains associated with the activation site and nucleates formation of the membrane attack complex. C5a engages specific G protein–coupled receptors such as C5aR1 on neutrophils, monocytes, macrophages, and other immune and non‑immune cells, where it triggers chemotaxis, degranulation, upregulation of adhesion molecules, oxidative burst, and release of inflammatory mediators, linking complement activation to cell recruitment, vascular permeability changes, and amplification of local and systemic inflammatory responses. C5b sequentially recruits C6, C7, C8, and multiple C9 molecules to assemble the C5b‑9 membrane attack complex that inserts into susceptible membranes and forms transmembrane pores, leading to osmotic imbalance and lytic death of bacteria and other complement‑sensitive targets, and contributing to clearance of damaged or altered host cells under certain conditions. Regulation of C5 activation and its effector functions is mediated by fluid‑phase and membrane‑bound inhibitors such as factor H–controlled upstream convertase regulation, vitronectin and clusterin binding to soluble C5b‑9, and CD59 on host cells that blocks C9 polymerization, collectively restricting terminal complex assembly and protecting host tissues from complement‑mediated damage while preserving antimicrobial efficacy. Functional roles of C5 and its fragments extend into modulation of adaptive immunity, as C5a–C5aR signaling influences dendritic‑cell maturation, T helper cell polarization, and B cell responses, integrating complement activation with shaping of antigen‑specific immunity and tolerance. Dysregulated C5 activation or impaired control of C5b‑9 assembly contributes to pathology in disorders such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, membranous nephropathies, age‑related macular degeneration, and a range of autoimmune and inflammatory diseases, where excessive terminal pathway activity drives hemolysis, endothelial injury, and tissue inflammation.

Background

Complement component C5 is a central effector protein of the complement cascade that links upstream C3 convertase activity to generation of potent anaphylatoxin and cytolytic responses, and belongs to the family of large modular complement proteins that circulate mainly as inactive precursors until they are cleaved by C5 convertases on activating surfaces. The molecule consists of two chains derived from a single precursor and organized into multiple β‑sheet‑rich domains homologous to those in C3 and other terminal complement components, creating surfaces that support recognition by C5 convertases and that, after cleavage, yield a soluble effector fragment and a membrane‑binding fragment that initiates terminal complex assembly. Conversion of C3 into C3b during classical, lectin, or alternative pathway activation allows assembly of pathway‑specific C5 convertases, which bind native C5 and cleave it into the small fragment C5a and the larger fragment C5b; C5a diffuses away and acts as a strong anaphylatoxin, whereas C5b remains associated with the activation site and nucleates formation of the membrane attack complex. C5a engages specific G protein–coupled receptors such as C5aR1 on neutrophils, monocytes, macrophages, and other immune and non‑immune cells, where it triggers chemotaxis, degranulation, upregulation of adhesion molecules, oxidative burst, and release of inflammatory mediators, linking complement activation to cell recruitment, vascular permeability changes, and amplification of local and systemic inflammatory responses. C5b sequentially recruits C6, C7, C8, and multiple C9 molecules to assemble the C5b‑9 membrane attack complex that inserts into susceptible membranes and forms transmembrane pores, leading to osmotic imbalance and lytic death of bacteria and other complement‑sensitive targets, and contributing to clearance of damaged or altered host cells under certain conditions. Regulation of C5 activation and its effector functions is mediated by fluid‑phase and membrane‑bound inhibitors such as factor H–controlled upstream convertase regulation, vitronectin and clusterin binding to soluble C5b‑9, and CD59 on host cells that blocks C9 polymerization, collectively restricting terminal complex assembly and protecting host tissues from complement‑mediated damage while preserving antimicrobial efficacy. Functional roles of C5 and its fragments extend into modulation of adaptive immunity, as C5a–C5aR signaling influences dendritic‑cell maturation, T helper cell polarization, and B cell responses, integrating complement activation with shaping of antigen‑specific immunity and tolerance. Dysregulated C5 activation or impaired control of C5b‑9 assembly contributes to pathology in disorders such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, membranous nephropathies, age‑related macular degeneration, and a range of autoimmune and inflammatory diseases, where excessive terminal pathway activity drives hemolysis, endothelial injury, and tissue inflammation.

References
[1] Kareem S, et al. Immunology. 2023 Oct;170(2):180-192. [2] Dunkelberger JR, et al. Cell Res. 2010 Jan;20(1):34-50.

PVDFメンブレン孔径参考表

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%