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Thread: Question Regarding Sandwhich ELISA

  1. #1 Question Regarding Sandwhich ELISA 
    New Member
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    Oct 2013
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    Hey everyone!
    I am currently developing a Sandwhich ELISA to help me detect miniscule levels of integrins (in a mixed sample). I just want to know whether the basic step is in the correct order (excluding the washing and blocking step which I have left out intentionally). Have I done the Sandwich ELISA in the correct order:
    1. Capture antibody
    2. Antigen containing sample
    3. Primary detection antibody
    4. Secondary HRP conjugated antibody – Anti-mouse IgG Biotinylated
    5. Extravidin peroxidase
    6. TMB + substrate-chromogen

    Im so confused whether or not the Anti-mouse IgG biotinylated should be HRP conjugated?
    I have read from Abcam datasheet recommends that 0.125g/ml of Biotinylated Anti-Mouse IgG should be used in ELISA, so I am planning on doing this in 2.5g in 20ml or is this too high? *confused.com*

    Thank you!!


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