We are performing an ELISA experiment to determine the binding of Complement antibodies against P. aeruginosa.
We have pNPP substrate (Thermo Scientific cat # 34045) instead of pNPP tablets.
We used 10mg substrate mixed in 10ml sterile distilled solution & after 30 mints still no change in color, what does that means?
We predicted two possible reasons, that may be 10mg/10ml pNPP substrate concentration is very low or may be we can't use sterile distilled & we should use Diethanolamine buffer. If anyone have used it please share. Thanks
I would use a buffer to make sure the pH is appropriate for the alkaline phosphatase detection enzyme.
pNPP is the substrate of phosphatase alkaline. The enzyme is usually linked to the detection antibody (or streptavidin...). This enzyme is active at an alkaline pH (pH > 7, hence the name alkaline). A straightforward way to check is to add a few microliters of the detection antibody (the solution used for the last incubation) to pNPP solution; an intense yellow colour should develop right away, otherwise adjust the pH as Adam B Shapiro suggested.
This excerpt might help [“The ELISA Guidebook” [ISBN 978-1-60327-254-4]:
"Alkaline Phosphatases are assayed in buffer depending on the source of the enzyme. For bacterial enzyme, 0.1 M Tris-HCl buffer, pH 8.1, containing 0.01% magnesium chloride is used. For intestinal mucosal enzyme a 10% (w/w) diethanolamine (97 mL in 1 L of a 0.01% magnesium chloride solution) buffer pH 9.8 (adjusted with HCl) is used. p -Nitrophenylphosphate (pnpp) is added just before use (available as preweighed pellets) to 1 mg/mL. The production of nitrophenol is measured at 405 nm.
The reaction is stopped by the addition of 0.1 volume of 2 M sodium carbonate. Note that inorganic phosphate has a strong inhibitory effect on AP and therefore PBS or similar buffers are to be avoided."
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