1: To create a reagent for your Western Blot, you purified an enzyme, horsereddi

Question

1: To create a reagent for your Western Blot, you purified an enzyme, horsereddish peroxidase from a horseradish (Armoracia rusticana) plant. Then, you run an enzyme kinetics assay on four increasingly pure fractions of your enzyme. For this kinetics assay, you used TMB, (3,3',5,5'-tetramethybenzidine) substrate that, after acidification, produced final diimine product adsorbing light at 450 nm with the molar absorptivity of E450nm=59,000 cm-1 M-1. You obtained V0 data using A450nm, and now need to calculate V0 in m/L. You used 100nM of TMB in a 96-well plate and the assay volume 200L that has a pathlength of 0.585cm. Using the Beer law, A=ebc, calculate initial velocity of each fraction in M/min.

2:You were able to obtain pure HRP enzyme and calibrate you kinetics assay. HRP concentrations that you obtained according to your calibration are listed in the table below (use V0 calculated in 1)

Based on the data in the table above, create a Lineweaver–Burk plot and estimate Vmax and Km of HRP from it.

1/V0 = Km/Vmax[S] + 1/Vmax Lineweaver–Burk equation *Use a separate sheet of paper to for your answer.

please please please help been going at this question for days.....

Explanation / Answer

There are several different types of HRP substrates. The type of substrate used for detection depends on the particular assay and mode of detection. Chromogenic HRP substrates, which remain in solution and become colored after reaction with HRP, are often used for ELISAs and other colorimetric assays. Commonly used chromogenic HRP substrates include 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2' -azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS). The choice of soluble chromogenic substrate depends on the requirements for assay sensitivity and the capabilities of the microplate reader.

HRP conjugates are commonly used in indirect assays. The horseradish peroxidase enzyme conjugate does not bind directly to the target but rather to an antibody or other molecule that has been bound to the target in a previous step. The indirect method has two advantages: the two-step procedure provides greater amplification because there are usually multiple binding sites for the conjugate on the molecule previously bound to the target, and the conjugate does not need to be specific for the target, but only for the molecule type bound to the target, saving time and money. For instance, an anti-mouse-HRP conjugate can be used in any assay where the primary antibody is mouse, or a protein A-HRP conjugate can be used for any biotinylated primary antibody.

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