explain the purpose of each of the control samples in the experiment(Effect of F

Question

explain the purpose of each of the control samples in the experiment(Effect of Forskolin on cAMP levels in HepG2 cell experiment)

Be sure to distinguish the functions of each of the controls from each other. Also include in your explanation specifically how the data would be compromised if each of the controls had not been run3

We made samples like summary chart to measure Forskolin samples. But we didn't directly measure optical density of Forskolin samples, but we measured controls together. The each component of controls is shown in the summary chart. And why did we need different controls?

a. Blank

b. TA

c. control

d. B0

Principle 1. Standards and samples are added to wells coated with a goat cu-rabbit lgG antibody. A solution of CAMP covalently conjugated to alkaline phosphatase is added, followed by a solution of rabbit polyclonal antibody to CAMP (ou-CAMPrabbit). 2. During a simultaneous incubation at room temperature the antibody binds, in a competitive manner, the CAMP in the sample or the CAMP conjugate. The plate is washed, leaving only o-CAMP-bound CAMP which will adhere to the well via interaction with the o-rabbit on the bottom of the well. 3. P-nitrophenylphosphate (pNpp) substrate solution is added. The substrate generates a yellow color (p-nitrophenolate, "pNp- when hydrolyzed by the alkaline phosphatase on the CAMP conjugate. 4. Stop solution is added. The yellow color is read at 405 nm. The amount of signal is inversely proportional to the amount of CAMP in the original sample. CAP CAP (from sample) CAME CAMP (from kit) AP (a rabbit IgG) add pNpp AP wash away unbound CAMP's Reagent Preparation 1. Wash Buffer Prepare the wash buffer by diluting 5.0 mL of the supplied Wash Buffer Concentrate with 95.0 mL of deionized water 2. CAMP Standards Allow the 2,000. pmol/mL standard stock to warm to room temperature

Explanation / Answer

1) Blank is a solution that doesnt contain things you are measuring whereas it act as base or give you zero reading. In spectrophotometer you have set a particular wavelength at which your anylate absorb most but sometimes there can be other things present in solution that absorb little of the same wavelength you are using and produce a false results. Here, in the blank we are adding substrate and stop solution after washing to check whether there is nonspecific activity due to plate itself. If blank solution producing colour means presence of contamination in plate cause non enzymatic hydrolysis of pNPP. If you will not add the blank solution you will get a variation in your results. Here, the plate itself to the measured OD, then you will get the false OD of your sample. Because you are getting a OD due to cAMP conjugate or cAMP and plate itself.

TA : This control is used to check enzymatic activity of cAMP-AP . Here, after washing we are adding cAMP-AP and pNPP substrate , so you will get the base line absorbance of enzymatic activity of AP. When cAMP-AP will bind to the antibody in microtiter plate, it will hydrolyse pNPP and gives yellow colour. This control is used to compare enzymatic activity in the presence of sample. Here, you can substract enzymatic activity in the presence of sample with in the absence of sample. So you will get a exact value. If this control is not added, then you cant say that whether the yellow colour appear due to the enzymatic activity of AP or due to other nonspecific activity.

Control : This will use to compare the difference between the amount of cAMP in the sample in the presence and absence of Forskolin treatment. Here, the amount of signal from control sample and treated sample will give you idea about amount of cAMP. This control is required to check the effect of Forskolin treatment. If this control is not present, you will not able to say whether forskolin stimulate changes in the cAMP production in cell.

B0 : This control contain all the reagents except sample. Using this solution you will get the absorbance without any competition with sample. Here, you will get maximum absorbance. This control is used to compare absorbance in the presence of competition with sample with the maximum absorbance. Using this you can get B/B0 means intensity of sample/ maximum intensity. If this control is not present then you will not get the comparison and you will not get the maximum limit of your values with sample. In that case there is a chance to get a false result.

We need different control for different purposes. In sandwich ELISA, B0 standard is necessary to get maximum intensity, control require to study the effect of forskolin treatment on HepG2 cells, AT and blank require to avoid nonspecific results. So, each control serve different purposes to finally get you a accurate values and good results. In this case single standard will not solve all the problems.

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