Bovine Flotillin-1 (FLOT1) ELISA Kit

This assay employs a two-site sandwich ELISA to quantitate FLOT1 in samples. An antibody specific for FLOT1 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any FLOT1 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for FLOT1 is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of FLOT1 bound in the initial step. The color development is stopped and the intensity of the color is measured.
 

Flot1 encodes a deduced 428-amino acid protein with a predicted molecular mass of 47 kD. It contains 2 hydrophobic domains and several potential phosphorylation sites. Flot1 shares 47% amino acid identity with both mouse and human FLOT2. Northern blot analysis of mouse tissues revealed expression in adipose tissue, heart, skeletal muscle, and lung. By Western blot analysis, Flot1 was also found as a 47-kD protein in caveolin-rich membrane domains isolated from human lung and from cultured mouse adipocytes. Flotillin-1 colocalized with PTOV1 at the plasma membrane and in the nucleus, and it entered the nucleus concomitant with PTOV1 shortly before initiation of S phase. Protein levels of PTOV1 and flotillin-1 oscillated during the cell cycle, with a peak in S phase.
 

Reagents	Quantity	Reagents	Quantity
Assay plate (96 Wells)	1	Instruction manual	1
Standard (lyophilized)	2	Sample Diluent	1 x 20 mL
Biotin-Conjugate (concentrate 100 x)	1 x 120 μL	Biotin-Conjugate Diluent	1 x 12 mL
Streptavidin-HRP  (concentrate 100 x)	1 x 120 μL	Streptavidin-HRP Diluent	1 x 12 mL
Wash Buffer (concentrate 25 x)	1 x 20 mL	Substrate Solution	1 x 10 mL
Stop Solution	1 x 6 mL	Adhesive Films	4

 

This assay has high sensitivity and excellent specificity for detection of Bovine FLOT1. No significant cross-reactivity or interference between Bovine FLOT1 and analogues was observed.
 

Matrices listed below were spiked with certain level of recombinant Bovine FLOT1 and the recovery rates were calculated by comparing the measured value to the expected amount of Bovine FLOT1 in samples.
 

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in forty separate assays to assess inter-assay precision. CV (%) = SD/meanX100 Intra-Assay: CV<8% Inter-Assay: CV<12%
 

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Bovine FLOT1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
 

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).
 

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
 

Store at 2-8°C. Please refer to Instruction Manual.