We did biochemical assays like esterase and phosphatase (quantitatively) activity in the whole body homogenate of the larval extract. For that, first we have to estimate the protein concentration of the whole body homogenate and based on this protein concentration , we have to estimate the esterase and phosphatase activity using larval whole body homogenate. But here the confusion was started that in protein concentration of the whole body homogenate of the control was in low compared to treated (high) but the esterase and phosphatase activity was reduced in treated when compared to control. How it was possible that while protein concentration of treated was high but the level of the enzyme was in low in treated when compared to control (enzyme level was higher).
You can do two things (and probably should do both):
- Compare the enzyme content per larva. Homogenise the animals so that 1 mL corresponds to a given number of animals, or a given animal weight.
- Compare the enzyme content based on protein. Dilute your homogenates so that protein content for all samples is, say, 1 mg/mL.
The answer you get may be different for these experiments. For example, a treatment may reduce the overall protein expression (reduced growth due to poisoning), but the expression of particular proteins may increase, if these proteins allow the organism to deal with your treatment. Think for example drug resistance transporters or CYP450 hydroxylases.
Normalizing is important -- per Englebert's suggestions above. The problem with normalizing is the 'normal' part. Whatever your are doing to these larvae seems to be changing a fairly universal parameter like total protein. It might be worth tracking both metrics mentioned above -- the individual's mass and total protein are both easy and reliable measurements. You might also try normalizing against the level of a specific 'house-keeping protein' -- some protein that you believe should be invariant to your treatment conditions. A lot of research papers that use Westerns to quantitate their proteins of interest use GAPDH as their normalization reference level -- stained on the same Westerns, and then the protein biomarkers are reported as a ratio. Not sure if GAPDH would be appropriate for your larvae. Perhaps another well-characterized larval protein instead?
Entirely separate thought: Have you considered whether or not the activity of the enzymes may be altered -- and NOT their level. Allosteric changes or other co-factor changes can alter activity without changing the amount/concentration of the enzyme. You might measure 'specific activity' to know more: measure enzyme activity as you have been, but also quantitate the amount of enzyme itself -- via ELISA, Western, quantiative LC/MS, etc.
I must recommend HPLC-photometric enzyme assay (please see files; BAQ BIN assay and J Chrom B Rat BIN LIP Km).
Further, protein determination should be performed by our HPLC-SEC-photometric protein assay method (please see files; Prot Deter Summary and HPLC-Surf-SEC protein determination method).
Furthermore, ELISA (Immunologic method using monomer IgG), Western, LC/MS, MS, PCR, NMR, Flow cytometry, Direct utilization of photometer such as MTT method and NanoDrop, and Scanning Electron Microscope/SEM and/or Transmission Electron Microscope/TEM are all non-quantitative, since these methods do not present the recovery values which is most important in Analytical Chemistry.
Therefore, I have firstly demonstrated that Fucoidan of Okinawa, Japan, is actively transported in humans by utilizing reliable HPLC-photometric method (please see file; JCB Fucoidan Transport). The active transport may be similar to enzyme reaction at the hydrophobic membrane component (please see file; Ala Transport).
Interesting Species differences has been found; i.e. Carbonyl cyanide m-chlorophenyl hydrazone/ClCCP is effected to aerobic bacteria (Bacillus subtilis), but this ClCCP is not effective to humans (please see again file; JCB Fucoidan Transport).
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone/FCCP is surely effective to humans (please see again file; JCB Fucan Transport).
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