Rat Complement C3/C3 ELISA Kit- High Sensitivity

Product Name:	Rat Complement C3/C3 ELISA Kit
Product Code:	RTFI00611
Size:	96 Assays
Target:	Rat C3
Alias:	C3
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	1.875ng/ml
Range:	3.125-200ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat C3 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat C3 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-105	98
EDTA plasma(n=5)	93-104	98
UFH plasma(n=5)	86-101	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat C3 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	87-103%	87-105%	85-100%
EDTA plasma(n=5)	82-101%	84-100%	87-99%
UFH plasma(n=5)	87-96%	81-99%	82-97%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P01026

UniProt Protein Function:	C3: C3 plays a central role in the activation of the complement system. Its processing by C3 convertase is the central reaction in both classical and alternative complement pathways. After activation C3b can bind covalently, via its reactive thioester, to cell surface carbohydrates or immune aggregates. Defects in C3 are the cause of complement component 3 deficiency (C3D). A rare defect of the complement classical pathway. Patients develop recurrent, severe, pyogenic infections because of ineffective opsonization of pathogens. Some patients may also develop autoimmune disorders, such as arthralgia and vasculitic rashes, lupus-like syndrome and membranoproliferative glomerulonephritis. Genetic variation in C3 is associated with susceptibility to age-related macular degeneration type 9 (ARMD9). ARMD is a multifactorial eye disease and the most common cause of irreversible vision loss in the developed world. In most patients, the disease is manifest as ophthalmoscopically visible yellowish accumulations of protein and lipid that lie beneath the retinal pigment epithelium and within an elastin- containing structure known as Bruch membrane. Defects in C3 are a cause of susceptibility to hemolytic uremic syndrome atypical type 5 (AHUS5). An atypical form of hemolytic uremic syndrome. It is a complex genetic disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, renal failure and absence of episodes of enterocolitis and diarrhea. In contrast to typical hemolytic uremic syndrome, atypical forms have a poorer prognosis, with higher death rates and frequent progression to end-stage renal disease. Susceptibility to the development of atypical hemolytic uremic syndrome can be conferred by mutations in various components of or regulatory factors in the complement cascade system. Other genes may play a role in modifying the phenotype. Increased levels of C3 and its cleavage product ASP, are associated with obesity, diabetes and coronary heart disease. Short-term endurance training reduces baseline ASP levels and subsequently fat storage.
UniProt Protein Details:	Protein type:Inhibitor; Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 9q12 Cellular Component: extracellular space; protein complex Molecular Function:C5L2 anaphylatoxin chemotactic receptor binding; cofactor binding; endopeptidase inhibitor activity; lipid binding; protein binding Biological Process: blood coagulation; chemotaxis; complement activation; complement activation, alternative pathway; complement activation, classical pathway; fatty acid metabolic process; inflammatory response; phagocytosis, engulfment; positive regulation of activation of membrane attack complex; positive regulation of angiogenesis; positive regulation of developmental growth; positive regulation of G-protein coupled receptor protein signaling pathway; positive regulation of phagocytosis; positive regulation of protein phosphorylation; positive regulation of type IIa hypersensitivity; response to estradiol; response to estrogen; response to glucocorticoid stimulus; response to magnesium ion; response to progesterone; tolerance induction
NCBI Summary:	putative complement component C3; likely involved in innate immune response [RGD, Feb 2006]
UniProt Code:	P01026
NCBI GenInfo Identifier:	158138561
NCBI Gene ID:	24232
NCBI Accession:	NP_058690.2
UniProt Secondary Accession:	P01026,Q9ET19, Q9QV57, Q9QV58,
UniProt Related Accession:	P01026
Molecular Weight:
NCBI Full Name:	complement C3
NCBI Synonym Full Names:	complement C3
NCBI Official Symbol:	C3
NCBI Protein Information:	complement C3
UniProt Protein Name:	Complement C3
UniProt Synonym Protein Names:	Complement C3 beta chainC3-beta-c; C3bc
Protein Family:	Complement C3
UniProt Gene Name:	C3

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37°C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum:	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum and assay promptly or aliquot and store the samples at-80°C. Avoid multiple freeze-thaw cycles.
Plasma:	Collect plasma using EDTA or heparin as an anti-coagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid:	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell Culture Supernatant:	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell Lysates:	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20°C.
Tissue Homogenates:	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenizein 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or-80°C.
Tissue Lysates:	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk:	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.