Hello!
I'm starting my Master's project soon, and I'm in the optimization/practice step.
I'm stimulating RAW264.7 cells (in a 24 well) with LPS for 18h.
I did a first experiment today: a western blot to detect TNF-a in the supernatant.
I collected the supernatant, I concentrated the proteins, and I loaded everything on the gel.
I didn't see any bands at 17kDa. But there were way bigger bands over 100 kDa.
So I've got 2 questions.
1. What could be the reasons why there were no bands at 17kDa? Was the number of cells too small? Should I use a 12 well or 6 well instead?
2. Now this will probably be a very dumb question. But for this kind of experiment, do people usually do ELISA + a confirmation with WB? Or just one of these? Also, could I just use FACS? Would it be better?
Thank you so much
Terrie Lb to answer your 1st question, i would say that before going to WB, you should have checked the protein quantity by Bradford or BCA. And to check the quality of protein you must have run the sample on SDS-PAGE followed by coomassie blue staining. If there are multiple bands and not smear, that means you should go further for WB.
coming to your 2nd question: You should use ELISA for cytokine detection from supernatent rather than WB. If you have a set of cytokines to be assayed then go for CBA or Luminex platform, which are much sensitive than ELISA.
For the detection of TNF from the sup, Elisa is the better way.
the amount is too low for the western blot.
Hello Terrie,
I completely agree with Junjie Shao, cytokines should be estimated by ELISA. The amount of cytokines released in the cell supernatant is usually very less, and ELISA is very sensitive and specific to quantify that amount.
All the best for your successful project.
Dear Terrie,
If you will screen multiple cytokines, you can prefer cytometric bead array (CBA).
Good luck
Ishak
ELISA is the best way to detection of TNF-alpha from the cell supernatant.
Western blot will work but the sample will be very low.
Terrie Lb to answer your 1st question, i would say that before going to WB, you should have checked the protein quantity by Bradford or BCA. And to check the quality of protein you must have run the sample on SDS-PAGE followed by coomassie blue staining. If there are multiple bands and not smear, that means you should go further for WB.
coming to your 2nd question: You should use ELISA for cytokine detection from supernatent rather than WB. If you have a set of cytokines to be assayed then go for CBA or Luminex platform, which are much sensitive than ELISA.
Dear Terrie, Western Blot is probably the cheapest but the most tricky methodology. WB is a semi-quantitative methodology because it mainly indicates presence/absence and somehow allows showing the expression of a protein normalized against control or the protein load. On the other hand, ELISA provides you a quantitative result showing you the amount of cytokine that is produced. Flow Cytometry could also be tricky but got the advantage that lives cells are used and offers a kind of "in situ" detection of the cytokine. If you have money and don't have much time I would recommend the solution suggested by Dr. Rocio Campos Vega since only two days are required using these tests, but only a few samples (usually just 2) can be assayed at the same time.
You should check several manufacturers: R&D offers several alternatives but usually, only 2 samples can be evaluated (control + sample). If you want more samples, I would suggest Abcam (more samples, less proteins). It depends on the amount of proteins you would like to evaluate and the inflammation pathway. Please check this article from a former lab mate (Dr. Candice Mazweski)
10.1093/carcin/bgaa008
Best regards
- Badges
- Science method