In my research, I need to detect HPV 16E6 protein in western blotting. However, I always failed to detect this protein in caski cell line in western blotting. The primary antibody for HPV 16E6 is the antibody of Santa-crus technology. I have tried more than 50 times using different lysis methods, including herpes lysis buffer (NP-40), Tris lysis buffere (N-40) and Triton lysis buffer. No band found in western blotting. Anybody can help me to resolve this problem? Thank you very much.
In our hands, antibodies from Santa Cruz to detect the expression of HPV-16 E6 protein did not work. Therefore, it is likely that your antibody does not work. Therefore, you may want to consider to detect the transcript levels by qPCR.
Hi Jinn-Li,
I also purchased a santa cruz anti-body however for the HPV-18 E6 [sc-1586] and trying to get the blot to work for Hela cells. I have also not had much luck after more than 5 months trying to optimise conditions for western blot, co-IP and FACS. I sent a full report to the supplier of my data and failed attempts and am waiting to see what they suggest.
Let me know if you receive anything your side and i'll keep you posted about what un folds my side!
If you have tried to optimize and are still getting no bands, I advise that you call Santa Cruz and ask for a different antibody if they have one, or a credit on your account. Then shop around for other HPV 16E6 antibodies. Look for products that include WB images and links to published papers to help you evaluate the quality. Also- are you certain of your cell line ID? Do you know where they came from and what work has been done on previous passages?
I have seen a couple of presentations by ArborVita, and discussed with people who have used the system for E6 detection. The data were convincing and people were keen. But I do not know whether you can use the Abs for WB, outside their kit for detection of active infection by oncogenic HPVs.
Have you confirm the presence of HPV DNA prior to carrying out the Western Blotting experiment? If yes, then we could argue whether your antibody is not working properly or the lysis method itself is not sufficient. I think the preliminary step (HPV DNA detection) is really important to know whether the HPV itself exist in your sample(s). Otherwise, you may run blind. :)
Dear Jinn-Li,
If you could not detect E6 protein using Santa cruz Ab try the following:
1. Have you checked the presence of B-Actin as an internal control in your samples?
2. Have you used positive control to check if you have done WB correctly?
3. If the answer for the previous questions is YES, so change your Ab and try to purchase a new one from another company.
4. If you could not find any, try targeting HPV16 E7 protein using WB!
5. Check your protein extraction method again!
Caski cell should have abundant amount of HPV (up to 600 copies). Try this protocol using extraction Buffer 2.5x (Tris 0.25M pH6.8, Glycerol 50%, SDS 10%) for cells in cell culture using the following procedure:
1. Grow freshly seeded cells for 24 hours to obtain 65 – 75 % confluency (corresponds for CaSki cells to a plating of approx. 2x106 cells/10cm Ø cell culture dish). Put cell culture dishes with medium on ice for 5 minutes.
2. Aspirate media from cultures and keep dishes on ice.
3. Wash cells two times with ice-cold PBS 1x (the ice cold PBS is of particular importance if kinase signaling studies are conducted).
4. Aspirate all remaining PBS with pasteur pipet (there will remain 50 – 200 µl of liquid as thin non-aspirable layer).
5. Put plates at room temperature and immediately lyse cells by the addition of warm Extraction Buffer (2.5x) preheated to 96oC, suggested volume is specified below. Immediately scrape the cells off the dish and transfer the extract to a microcentrifuge tube.
6. Sonicate 2 x 10 sec to complete cell lysis and shear DNA (to reduce sample viscosity) at room temperature. Do not put samples on ice (extraction buffer contains SDS).
7. Heat the sample at 96°C for 10 min.
8. Microcentrifuge for 5 min at > 12000g at room temperature.
9. Transfer the supernatant into a new tube.
10. Add DTT to final concentration 100 mM. Mix well.
11. Heat the sample at 96°C for 10 min.
12. Load 10 or 20 µl onto SDS-PAGE gel
Good luck!
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