Testing Data
C4d complement Antibody | anti-C4A antibody
Anti - C4d complement
1. Deparaffinize the section in 3 changes of xylene, 10 minutes each.
2. Wash the section in 96%, 80% and 70% ethyl alcohol for 10 minutes each.
3. Rinse in distilled water, 2 x 5 minutes.
4. Block the endogenous peroxidase by incubating the tissue in 3% hydrogen peroxide (H2O2) for 10 minutes.
5. Wash in distilled water, 2 x 5 minutes.
6. For antigen retrieval: immerse the slide in Tris-EDTA buffer, pH 9.0*, and incubate in water bath for 20-25 minutes at 96-98°C.
7. Remove the staining to room temperature and let the slide to cool (in Tris-EDTA buffer, pH 9.0) for 20 minutes.
8. Rinse in distilled water, 2 x 5 minutes.
9. Wash in 0.05 M Tris-HCl, pH 7.6 buffer supplemented with 0.2% of Tween-20 or PBS buffer supplemented with 0.2% of Tween-20 (buffer A), 2 x 5 minutes.
10. Concentrated:
Incubate the section with primary antibody at the dilution 1:100 - 1:200 for 1 hour in the closed wet chamber.
Ready To Use (RTU):
Incubate the section with primary antibody (ready to use) for 1 hour in a closed wet chamber.
11. Wash 3 x 5 minutes with buffer A.
12. Apply the secondary antibody, and proceed to standard immunohistochemistry protocol (HRP - Peroxide - DAB). (Please Inquire).
13. Wash 3 x 5 minutes with buffer A.
14. Apply the chromogen (DAB), 1 - 3 minutes.
15. Wash in distilled water, 2 x 5 minutes.
16. Rinse in solution CuSO4 . 5H2O /0.90g NaCl + 0.50g CuSO4.5H2O in 100ml distelled water/.
17. Wash in distilled water, 1 x 2 minutes.
18. Stain in hematoxylin for 5 minutes.
19. Wash in distilled water, 3 x 2 minutes.
20. Rinse in 37mM amonium hydroxide solution.
21. Wash in distilled water, 1 x 2 minutes.
22. Mount the slide for observation.
Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA solution, 0.05% Tween-20, pH 9.0):
Tris ----------- 1.21 g; EDTA ---------- 0.37 g; Distilled water ------------ 1000 ml Mix to dissolve in 700 ml of distilled water. Adjust pH to 9.0 with 1M HCl and then add 0.5 ml of Tween-20 and mix well. Adjust the final volume to 1 liter with distilled water. Store this solution at room temperature for 3 months or at +4ºC for longer storage.
Short application protocol for Ventana Benchmark Slide Staining System
1. Deparafinization.
2. Heating (72°C) at the medium temperatures. Deparafinization.
3. Cell conditioning (Enter).
4. ULTRA conditioner # 1 (Enter).
5. Heating glass (95°C), incubation 8 min. (Cell conditioner #1).
6. ULTRA CC1 solution application - 20 min. (Enter).
7. Titration (Enter).
8. Hand apply - primary antibody. Incubation 36 min. - dilution: 1:100, 1:200.
9. Nuclear stain (Enter).
10. Hematoxylin II application - one drop (nuclear stain). Cover and incubate 12 min.
11. After nuclear stain (Enter).
12. Bluing reagent application, one drop. After nuclear stain, cover and incubate 4 min.
2. Centrifuge the vial before use.
3. Intended for professional In Vitro identification use in laboratories.
4. Do not use after expiration date stamped on vial label.
5. Avoid contamination of the reagent.
6. Any discrepancies in the recommended procedures stated in the working protocol may affect the final results.
7. The reagent contains sodium azide (NaN3) which is highly toxic in higher concentrations. The concentration in the reagent (0.05%) is not considered as hazardous.
8. Disposal of waste material must be conducted in accordance with local regulations.
9. Wear appropriate Personal Protective Equipment to avoid contact with eyes and skin.
Testing Data
Testing Data
Testing Data #2
Testing Data #2
NCBI and Uniprot Product Information
NCBI Description
This gene encodes the acidic form of complement factor 4, part of the classical activation pathway. The protein is expressed as a single chain precursor which is proteolytically cleaved into a trimer of alpha, beta, and gamma chains prior to secretion. The trimer provides a surface for interaction between the antigen-antibody complex and other complement components. The alpha chain may be cleaved to release C4 anaphylatoxin, a mediator of local inflammation. Deficiency of this protein is associated with systemic lupus erythematosus and type I diabetes mellitus. This gene localizes to the major histocompatibility complex (MHC) class III region on chromosome 6. Varying haplotypes of this gene cluster exist, such that individuals may have 1, 2, or 3 copies of this gene. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Nov 2011]
Uniprot Description
Function: Non-enzymatic component of C3 and C5 convertases and thus essential for the propagation of the classical complement pathway. Covalently binds to immunoglobulins and immune complexes and enhances the solubilization of immune aggregates and the clearance of IC through CR1 on erythrocytes. C4A isotype is responsible for effective binding to form amide bonds with immune aggregates or protein antigens, while C4B isotype catalyzes the transacylation of the thioester carbonyl group to form ester bonds with carbohydrate antigens. Ref.19 Ref.21Derived from proteolytic degradation of complement C4, C4a anaphylatoxin is a mediator of local inflammatory process. It induces the contraction of smooth muscle, increases vascular permeability and causes histamine release from mast cells and basophilic leukocytes. Ref.19 Ref.21
Subunit structure: Circulates in blood as a disulfide-linked trimer of an alpha, beta and gamma chain.
Subcellular location: Secreted.
Tissue specificity: Complement component C4 is expressed at highest levels in the liver, at moderate levels in the adrenal cortex, adrenal medulla, thyroid gland,and the kidney, and at lowest levels in the heart, ovary, small intestine, thymus, pancreas and spleen. The extra-hepatic sites of expression may be important for the local protection and inflammatory response. Ref.23
Post-translational modification: Prior to secretion, the single-chain precursor is enzymatically cleaved to yield non-identical chains alpha, beta and gamma. During activation, the alpha chain is cleaved by C1 into C4a and C4b, and C4b stays linked to the beta and gamma chains. Further degradation of C4b by C1 into the inactive fragments C4c and C4d blocks the generation of C3 convertase. The proteolytic cleavages often are incomplete so that many structural forms can be found in plasma.N- and O-glycosylated. O-glycosylated with a core 1 or possibly core 8 glycan. Ref.24 Ref.31 Ref.33
Polymorphism: The complement component C4 is the most polymorphic protein of the complement system. It is the product of 2 closely linked and highly homologous genes, C4A and C4B. Once polymorphic variation is discounted, the 2 isotypes differ by only 4 amino acids at positions 1120-1125: PCPVLD for C4A and LSPVIH for C4B. The 2 isotypes bear several antigenic determinants defining Chido/Rodgers blood group system [
MIM:614374]. Rodgers determinants are generally associated with C4A allotypes, and Chido with C4B. Variations at these loci involve not only nucleotide polymorphisms, but also gene number and gene size. Some individuals may lack either C4A, or C4B gene. Partial deficiency of C4A or C4B is the most commonly inherited immune deficiency known in humans with a combined frequency over 31% in the normal Caucasian population (Ref.23). C4A6 allotype is deficient in hemolytic activity. Allotype C4A13 is infrequent.
Involvement in disease: Complement component 4A deficiency (C4AD) [MIM:614380]: A rare defect of the complement classical pathway associated with the development of autoimmune disorders, mainly systemic lupus with or without associated glomerulonephritis.Note: The disease is caused by mutations affecting the gene represented in this entry. Ref.20Systemic lupus erythematosus (SLE) [MIM:152700]: A chronic, relapsing, inflammatory, and often febrile multisystemic disorder of connective tissue, characterized principally by involvement of the skin, joints, kidneys and serosal membranes. It is of unknown etiology, but is thought to represent a failure of the regulatory mechanisms of the autoimmune system. The disease is marked by a wide range of system dysfunctions, an elevated erythrocyte sedimentation rate, and the formation of LE cells in the blood or bone marrow.Note: Disease susceptibility is associated with variations affecting the gene represented in this entry. Interindividual copy-number variation (CNV) of complement component C4 and associated polymorphisms result in different susceptibilities to SLE. The risk of SLE susceptibility has been shown to be significantly increased among subjects with only two copies of total C4. A high copy number is a protective factor against SLE. Ref.22 Ref.28
Sequence similarities: Contains 1 anaphylatoxin-like domain.Contains 1 NTR domain.
Sequence caution: The sequence AAB59537.1 differs from that shown. Reason: During cDNA synthesis, the 5' end has been inverted (Ref.6).The sequence BAE06071.1 differs from that shown. Reason: Erroneous initiation. Translation N-terminally shortened.
Research Articles on C4A
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Product Notes
The C4A c4a (Catalog #AAA684029) is an Antibody and is intended for research purposes only. The product is available for immediate purchase. AAA Biotech's C4d complement can be used in a range of immunoassay formats including, but not limited to, IHC-P: 1:100 - 1:200. Immuohistochemistry-Paraffin (IHC-P). Researchers should empirically determine the suitability of the C4A c4a for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "C4d complement, Antibody" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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