I have isolated a protein from algae and want to subjected it for anti-cancer activity
Dear Kishore,
The first step is cell viability studies in vitro. You culture the cancer cell lines and treat them with different concentrations of your protein. Then the measure of cell death or viability could be determined via MTT test. Through this test you would determine the IC50 (50% Inhibition Concentration, namely the protein concentration in which 50% of the cells are killed or inhibited from growth). If IC50 is rational and reasonable, your protein might be a drug candidate for cancer therapy!
http://en.wikipedia.org/wiki/MTT_assay
Cheers!
Hi Kishore,
First, what type of cancer would you like your algae be tested? If you would like, you may want to try HeLa cells, one of the most common cancer cell line, and MCF-7, a breast cancer cell line. Usually, you would want to know if your natural compound could inhibit growth or proliferation of these cancer cells. However, you should start determining the IC50 of your compound in healthy cell lines first. In this way, you'll be able to know what concentration of your compound you would use. Ideally, at that particular concentration, the healthy cells continue to grow and proliferate while cancer cells are inhibited. I hope the following outline will help you:
Cytotoxicity Assay
1. Determine water solubility of your compound. This will be helpful in solubilizing your compound in culture medium. If insoluble in water, you have to decide if what solvent can be used e.g., DMSO, ethanol, that suits your protein without losing activity
2. Find an available healthy cell line that originated from a similar tissue where the cancer line was derived e.g., HeLa cells = cervical cancer cell line, so I hope you find a healthy cervical cell line (most likely a primary cell line)
3. At various concentration of your protein, usually at 10-fold dilution, treat your healthy monolayer of cells with your compound. Include untreated (negative) control or vehicle control (for compound that was water insoluble). *well plates Highest concentration may start at the max amount of your compound soluble in your medium. This is arbitrary.
4. Monitor cell proliferation (under microscope) and cell viability (MTT assay). You may want to try RealGlo from Promega, where this reagent can be added from seeding the cultures and will develop color over time if cells proliferate. Reagent was claimed not be cytotoxic. This can be measured by spectrophotometer plate reader.
4. Generate curves by plotting absorbance values. Determine IC50 and the concentration of compound where your cells proliferate well, or with least cytotoxic effect. Compare with your control.
5. Use this concentration of your compound to start with. You may now do another round of culture this time with the cancer cell line of interest. You may follow the steps provided with modifications e.g., cancer cell instead of healthy cells, highest concentration to start with = the least cytotoxic concentration, inclusion of anti-cancer compounds as positive controls, etc.
I hope this outline would help you in your experiment. All the best!
thank you sir, i will try you protocols and follow your suggestions. thank you once again
Hello,
To check anticancer activity of a compound first of all you have to do invitro cell viability assay. You have to culture most widely used cancer cell line like HeLa cell line and treat it with different concentration of your protein. Then measure cell viability count by MTT assay. You may reconfirm it by Neutral red assay. Then you have to determine the IC50. If your IC50 is significant then you can also do invivo assay.
In invivo assay you can use xenograft model in immunocmpromised mice and treat the animal with your protein and check if there is any reduction of tumor volume. If your test is statistically significant, then you can reconfirm that your compound has some anticancer activity.
All the best.
- Badges
- Science topic
- Similar topics
- Geoscience
- Asia
- India