We have a plant where in the literature says it has insulin mimicking peptide. So we established callus from the leaf and the stem. Now we have to go further to identify the presence of that peptide in the callus cultures. Do anybody have idea or protocal
Hello Raghavendra,
I can think of the following means:
1. Butanol extractions of fresh callus (do not dry; may lyophilize) would help. Butanol: aqueous (1:1) or other rations, extractions help too. Instead of going to step 2, directly perform step 3 with "crude extracts" first- and if the results are negative in step 3 or 3A, then need to change your plant material or plant part/ tissue-type.
2. Run preparative TLC or better HPLC to resolve/ separate the peptides based on fractions. Stain fractions on TLC plates or in cuvettes (spectrophotometry) for "Ninhydrin" staining for 'peptides'. Save in parallel; the bands (from TLC) or fractions (from HPLC) for peptide -based bioassays in step 3.
3. Take all +vely stained fractions/ bands for mouse-based bioassays in streptozotocin- or alloxan-induced mice to show antihyperglycemic potential, restoration of beta cell functions, insulin resistance status, using ELISAs, histochemistry, with 'insulin' as positive control, and other anti diabetic drugs as positive controls (metformin to BGs as well).
4. Send all fractions that restore beta-cell functions, insulin status, blood sugar levels - if evident from 100s of such mice or rat based assays, can have potential "insulin mimicking" properties.
5. Using MALDI-ToF or LC-ESI-MS/MS obtain the structure/ chemistry of the fractions/ bands that were active in bio-assays to see if they are "peptides" or not.
6. If steps 3 and 5 work "positive" for you, then they are "insulin-mimicking peptides".
These are the "ideas". You need to find detailed protocols from 100s of papers available in the internet.
Note: Step 3.A: Instead of mice and rats, you can choose to use "cell lines" for glucose -uptake assay inhibition etc., using 13 C labeled glucose.
Warning: Laborious project, very expensive, but if you find results they very useful for lot of applications.
Question is: Why do you expect insulin-mimicking peptides to be present in callus and not the leaf and stem? If you get enough leaf and stem, then do it- because callus is expensive, needs sophistication and useless for pharmaceutical industry to invest so much for generating the raw materials.
Do not be surprized to find out at the end that the fractions that were active was not a peptide but a 'small molecule such as flavonoid, polyphenol terpenoid or a glycan among others'.
If you work hard and plan well, can be doable in a good 6 months period of time- of 100% effort. Nothing less.
If callus or leaf or stem, start with "1-10s of kilograms" of starting materials to be on safe side- most often we start with 1-100 grams and not enough left till you reach step 6. Work closely with "chemists" [spectroscopist, spectrometrists] and "biologists" [animal handling, cell line experts, biochemists] to get anything done at all.
Thanks and all the very best,
Biswa
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