Rabbit Type I Collagen Detection Kit

Rabbit Type I Collagen Detection Kit - Cat Number: 6016 From Chondrex.

Research Field: ECM

Clonality: N/A

Cross-Reactivity:

Host Origin: N/A

Applications: N/A

Isotype: N/A

Detection Range: 10 µg/ml-0.16 µg/ml

Sample Type: Culture Media, Solubilized Collagen

Concentration: N/A

Immunogen:

DESCRIPTION: ELISA kits to quantify Type I Collagen

6012: Mouse Type I Collagen Detection ELISA Kit
6013: Rat Type I Collagen Detection ELISA Kit
6014: Bovine Type I Collagen Detection ELISA Kit
6015: Porcine Type I Collagen Detection ELISA Kit
6016: Rabbit Type I Collagen Detection ELISA Kit
6019: Canine Type I Collagen Detection ELISA Kit
6021: Human Type I Collagen Detection ELISA Kit
FORMAT: 96-well ELISA plate with removeable strips
ASSAY TYPE: Sandwich ELISA
ASSAY TIME: 5.5 hours
STANDARD RANGE: 6012/6013/6015/6021: 5 µg/ml to 0.08 µg/ml
6014/6016/6019: 10 µg/ml to 0.16 µg/ml
NUMBER OF SAMPLES: Up to 40 (duplicate) samples/plate
SAMPLE TYPES: Solubilized collagen and cell culture media
RECOMMENDED SAMPLE DILUTIONS: 1:1 - 1:1000
CHROMOGEN: OPD (read at 490 nm)
STORAGE: -20°C for 12 months
VALIDATION DATA: 6021 (only): Intra-assay (1.4-3.9%)/Inter-assay (2-6.6%)/Spiking Test (99-106%)
NOTES: These kits are intended to detect native type I collagen and will only weakly detect denatured
collagen

INTRODUCTION

Type I collagen is one of several interstitial fibrillar collagens consisting of two identical α1(I) chains and one α2(I) chain. It is the mostabundant collagen type and is found in most connective tissues such as skin, bone, tendon, ligament, and heart. Many tissues containheterotypic fibrils meaning that two or more distinct collagen types coexist. For example, most connective tissues (except for bone) containheterotypic fibrils of type I and III collagen, although the type III collagen content is minor compared to Type I collagen. Chondrex, Inc.provides a variety of species-specific Type I Collagen Detection ELISA Kits designed to quantify the amount of species-specific type I collagenin cultured cells and tissue specimens.

ANTIBODY SPECIFICITY 

Monoclonal antibodies are used as the capture and detection antibodies in these Type I Collagen Detection ELISA Kits. All clones are highly specific to the native conformation of their respective type I collagens and poorly cross-react with denatured type I collagen. Chondrex, Inc. recommends carefully selecting kits to determine species-specific type I collagen in samples containing other species of type I collagen or type II collagen based on the specificity of the capture antibodies in the table below. Some capture antibodies are highly specific to their respective type I collagens and do not cross-react with other species of type I or type II collagen. 

KIT COMPONENTS

ASSAY OUTLINE

PLATE MAPPING

SOLUBILIZING COLLAGEN

For determining collagen content in cultured cell layers and tissues by ELISA, solubilizing collagen is required. For tissue specimens, a limited digestion with pepsin is highly recommended for native collagen preparation as other neutral proteinases, such as pronase and papain, digest collagen into small peptides. Pepsin only digests telopeptides located on both the N- and C-terminals of the collagen molecule and is not capable of digesting the helical conformation region of the collagen molecule itself. Please inquire with Chondrex, Inc. customer service for “Tips on Solubilization of Collagen”.

Solubilizing collagen from tissues by limited pepsin digestion (generally collagen to pepsin ratio is 100:1) depends on the types of tissues and the contents of the intra- and inter-molecular cross-linkages. For example, bone and Achilles tendon are resistant to pepsin digestion and only 10-20% of the collagen tissue will be solubilized. Young calf skin collagen will be completely solubilized by pepsin digestion within 24-48 hours, while it takes 7-9 days to solubilize adult skin. Pepsin resistant insoluble collagen might be solubilized by alkaline treatment. Suspend insoluble collagen in cold 0.1N NaOH solution containing 10% Na2SO4 and 0.1M Amine such as Tris and incubate at 4°C for 1-2 weeks. After treatment of collagen with alkaline, neutralize the pH to 5.0 with HCl, and then dilute it with 0.05M acetic acid or neutral buffer  such as 0.1M Tris-0.3M NaCl, pH 7.5

Therefore, the optimum solubilization condition for individual samples should be determined before processing samples. Collagen can be analyzed by 6% SDS-gel under non-reducing conditions, using authentic type I collagen as a standard. If samples contain bands larger than the -chain (MW = 300 Kd), the samples must be further digested by pepsin or elastase which will digest the intra- and inter-collagen crosslinkages. On the other hand, if smaller bands or smear bands are observed under the α-chain (MW = 100 Kd), the samples might be overdigested. Therefore, it is critical to understand the biological and physico-chemical properties of individual collagen samples.

NOTES BEFORE USING ASSAY

NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Crystals may form in Wash Buffer, 20X when stored at cold temperatures. If crystals have formed, warm the wash buffer by placing the bottle in warm water until crystals are completely dissolved.
NOTE 4: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 5: Cover the plate with plastic wrap or a plate sealer after each step to prevent evaporation from the outside wells of the plate.
NOTE 6: For partial reagent use, please see the assay protocol’s corresponding step for the appropriate dilution ratio. For example, if the protocol dilutes 50 µl of a stock solution in 10 ml of buffer for 12 strips, then for 6 strips, dilute 25 µl of the stock solution in 5 ml of buffer. Partially used stock reagents may be kept in their original vials and stored at -20⁰C for use in a future assay.
NOTE 7: This kit contains animal components from non-infectious animals and should be treated as potential biohazards in use and for disposal.