|
SampleTypes |
|
cell culture supernatant, urine
|
|
Specificity |
|
Designed for detection of mouse albumin in cell culture supernatants and urine
|
|
Protocol |
|
• Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-30°C).
• Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator.
• Add 50 μl of standard or sample per well, and cover wells and incubate for 1 hour. Start the timer after the last sample addition.
• Wash five times with 200 μl of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to complete remove liquid at each step.
• Add 50 μl of Biotinylated Rat Albumin Antibody to each well and incubate for 30 minutes.
• Wash five times with 200 μl of Wash Buffer.
• Add 50 μl of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance.
• Wash five times with 200 μl of Wash Buffer.
• Add 50 μl of Chromogen Substrate per well and incubate for about 10 minutes or till the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip.
• Add 50 μl of Stop Solution to each well. The color will change from blue to yellow.
• Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. Please note that after the reaction is stopped for about 10 minutes, some black particles may be generated at high concentration point, which will reduce the readings.
|
|
Storage |
|
Store unopened kit at 2-8°C up to expiration date
|
|
References |
|
(1) Gekle M. (2004) Annu Rev Physiol.
(2) Schindler C et al. (1999) J Hepatol. 31(6):1132
(3) Hemmelder MH et al. (1997) Nephrol Dial Transplant. 12 Suppl 2:57-62
(4) Sesmilo G et al. (2004) Ann Intern Med. 133(2):111-22
(5) Wettstein R et al. (2004) Shock. 22(4):351-357
(6) Saito T et al. (1991) Jpn J Surg. 21(4):402-11
(7) Strand TA (2004) Am J Clin Nutr. 79(3):451-6
|
|
Contents/Specifications |
|
• Mouse Albumin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against mouse albumin.
• Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay.
• Mouse Albumin Standard: Mouse Albumin in a buffered protein base (800 ng, lyophilized).
• Biotinylated Mouse Albumin Antibody (70x): A 70-fold concentrated biotinylated
polyclonal antibody against mouse Albumin (120 ul).
• MIX Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml).
• Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml).
• Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90 ul).
• Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate
tetramethylbenzidine (8 ml).
• Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
|
|
Notes |
|
Sample Collection, Preparation and Storage
• Cell Culture Supernatants: Centrifuge cell culture media at 3, 000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles.
• Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes and assay. Dilute samples 1:100 into MIX Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.
|