Human ZAG PharmaGenie ELISA Kit (SBRS0182)

1. Pre-Coated 96-well Strip Microplate
2. Wash Buffer
3. Standard Peptide
4. Assay Diluent(s)
5. Biotinylated Peptide
6. HRP-Streptavidin
7. TMB One-Step Substrate
8. Stop Solution
9. Assay Diagram
10. Positive Control Sample
11. Capture Antibody
12. Technical Manual

Other materials and equipment required:

The Assay Genie Human ZAG PharmaGenie ELISA Kit (SBRS0182) will require other equipment and materials to carry out the assay. Please see list below for further details.

• Distilled or deionized water
• Precision pipettes to deliver 2 ul to 1 ml volumes
• Adjustable 1-25 ml pipettes for reagent preparation
• 100 ml and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Orbital shaker
• Aluminum foil
• Saran Wrap
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• SigmaPlot software (or other software that can perform four-parameter logistic regression models)

1. Prepare all reagents, samples and standards as instructed.
2. Add 100 ul detection antibody to each well.
3. Incubate 1.5 h at RT or O/N at 4°C.
4. Add 100 ul standard or sample to each well.
5. Incubate 2.5 h at RT.
6. Add 100 ul prepared streptavidin solution.
7. Incubate 45 min at RT.
8. Add 100 ul TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 ul Stop Solution to each well.
11. Read plate at 450 nm immediately.

ZAG is a 41 kDa soluble glycoprotein, which is a homologue of the class 1 major histocompatibility complex (MHC) heavy chain. ZAG was found to stimulate lipolysis through stimulation of adenylyl cyclase in a GTP-dependent process. ZAG produced weight loss when administered to obese mice without a reduction in food and water intake. Loss of body weight was entirely due to loss of adipose tissue with no effect on lean body mass. There was a three-fold increase in oxygen consumption by interscapular brown adipose tissue (BAT), suggesting an increase in thermogenesis. Recent studies have reported that ZAG is not only capable of stimulating lipolysis through activation of HSL, but also increases the utilization of the released NEFA through an increased level of UCPs in BAT and skeletal muscle.