DESCRIPTION

CKAP4 (Cytoskeleton-associated protein 4), also known as CLIMP-63 (cytoskeleton-linking membrane protein 63), is a 63 kDa cytoskeleton-linking membrane protein. CKAP4 is a novel DKK1-binding protein on the cell surface membrane of Madin-Darby canine kidney (MDCK) epithelial cells, and evaluated CKAP4-mediated DKK1 signaling in cancer cell proliferation. In addition, CKAP4 was modified with palmitate at Cys100 by palmitoyl acyltransferase DHHC2, and palmitoylation was required for CKAP4 localization to PM lipid rafts to promote cancer cell proliferation.

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the solution yellow. The color intensity is proportional to the quantity of bound protein, which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	2,285.1	76.5	3.3
2	8	531.0	33.0	6.2
3	8	208.5	6.6	3.2

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	2,274.9	86.7	3.8
2	16	559.2	38.4	6.9
3	16	204.6	11.8	5.8

RECOVERY

The recovery of human CKAP4 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:2	101	80-130
Cell lysate	1:160	97	94-104
	1:320	100	97-103

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The cell lysate was initially diluted 1:10.)

		Human serum(Sample diluent PT 3)	Cell lysate(Sample diluent PT 3-ec)
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	110	98
	Range (%)	92-129	89-106
1:8	Average% of Expected	94	126
	Range (%)	86-103	124-127
1:16	Average% of Expected	112	118
	Range (%)	97-127	110-127

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human CKAP4 produced at Sunlong Medical™.