Question

PRE-LAB PROTOCOL: Answer the following pre-lab questions: 1. 2. 4. How is mouse anti-goat lgG antibody generated? Why must sodium azide not be included in the secondary antibody incubation steps? Assuming that the overall yield of LDH enzyme in your prep was exactly 100%, how should the intensity of the LDH band in the NADH pool sample compare to that of the crude homogenate sample in your immunoblot? Remember to take into account the total volumes of NADH pool and crude homogenate and any dilutions done and show any calculations.

this is western blotting experiment and I need your help with questions. I will send the summary for this exiperiment

Answer 1

1. Mouse anti-goat IgG is produced by injecting whole IgG from goat serum into mice. The antibody can be produced by hybridoma technology. B cells and myeloma cells are fused and then selected on HAT medium. The clone of hybridoma cells that produce the right antibody is selected by ELISA and the clone is then expanded to produced more antibodies. The antibodies can then be conjugated to Horse radish peroxidase (HRP) enzyme.

2. The secondary antibody is conjugated HRP enzyme for western blot. However, HRP is inhibited/deactivated by sodium azide. Hence, sodium azide should not be added to secondary antibody steps. Otherwise, no colorimetric detection can occur.

4. Lactate dehydrogenase is an enzyme that is involved in the interconversion of pyruvate and lactate. There is an associated conversion of NADH to NAD+. In the crude homogenate, LDH is not concentrated. Hence, amount of LDH in this homogenate is dilute. When the NADH pool sample is used, LDH is concentration in a small volume. Hence, LDH band in the NADH pool sample should be brighter or more intense the LDH band in crude homogenate. The crude homogenate should show a faint band of LDH.

The volumes used to dissolve the purified LDH from NADH pool as well as the volume of crude homogenate is not known. Further, dilution used and volume of sample loaded on gel is not known. Hence, calculation cannot be performed.