Application
ELISA
Sample
Human Recombinant protein (St. protein from Peprotech ELISA development kit ,)
Specification
St. protein from Peprotech ELISA development kit ,
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Type
Sandwich (Capture)
Other product details
Incubation time
1 hour(s) and 0 minute(s) · Diluent: PBS pH 7.4
Dilution
1/400
Secondary antibody
Secondary antibody
None used
Additional data
Additional Notes
Sandwich ELISA was done in order to test dose ab124962 works in pair with ab25074 or not.
High binding ELISA plate was coated with ab 124962 in concentrations 2.5, 5.0 and 10.0 ug/ml ( 400X, 200X and 100X dilution rate correspondently), plate was blocked with 1%BSA solution. Antigen - recombinant IL-1RA protein form Peprotech kit was added in concentrations 10.0, 1.0 and 0.1 ng/ml using diluent (1%BSA, 0.05%Tween-20, PBS) and incubated for 1h at RT.
Biotinilated Anti-IL-1RA a b25074 was used as detection antibody in concentrations 0.5, 0.25 and 0.125 ug/ml ( 1000X, 2000X and 4000X dilution rate correspondently ), plate was incubated with detection antibody for 1h at RT. After wash step, plate was incubated with Streptavidine solution 1: 200 (R&D) for 20min at RT for colour reaction development TMB solution was used. Plate was incubated for 15 min at RT in dark and stopped using 1M H2SO4 solution. Optical density was read using ELISA reader at 450nm.
We conclude that ab124962 and ab25074 don't work in pair in Sandwich ELISA, more probably they recognise the same epitope of antigen.
Abcam response
The antibodies tested by customer aren't tested as pair in sELISA. We recommend customers to use tested pair of antibodies for this application.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
MR. Toomas Neuman
Verified customer
提交于 Jan 29 2014