A. Cell : IEC-6 (rat epithelium from duodenum)

-------------------------------------

B. I treated the cells in 4 treatments

-Negative Control: Cultured in normal incubator (21% O2) with DMEM media.

-Treatment: Cultured in hypoxia incubator (1% O2) with DMEM media for 3 and 24 hours.

-Positive Control: Cultured in normal incubator (21% O2) with DMEM media + 100 uM CoCl2.

-------------------------------------

C. Protocol

Lysis buffer I used:

- 990 uL RIPA buffer (Thermo fisher) + 10 uL protease inhibitor complex

- 990 uL RIPA buffer (Thermo fisher) + 10 uL protease inhibitor complex + CoCl2 (final concentration 1 mM)

- 1X Laemli buffer with mercaptoethanol

Primary antibody: HIF-1A (D2U3T) Rabbit mAB #14179 by Cell Signaling Technology

Expected target size: 120 kDa

This is my extraction protocol:

1. 0.3 X 10^6 IEC-6 cultured in 6 well plates, with 2 mL media (DMEM + 10% FBS, 1% A/A & 1% Glutamax). Until confluency is reached (24H)

2. Before treatment wash the cells 2X with DPBS, to get rid of the FBS.

3. Add the media and culture as described above.

4. Take out the sample, and put it on an ice tray.

5. Wash the cells 2X with ice-cold DPBS.

6. Add 150 uL of lysis buffer scraped the cell and put into a microtube

For the RIPA buffer,

I incubated it for 10 minutes on ice and vortex during the 5th and 10th minute mark. Then, I centrifuged for 14,000 xg for 10 minutes at 4C. Then, I separated the supernatant from the pellet. The sample is stored at -20C for western blot (2 days after extraction). On the day of the western blot, I dilute it with Laemmli buffer and boil the sample at 95C for 5 minutes. Then, I proceeded to the western blot.

For the Laemlli buffer,

After scraping vortex, boiled at 95C for 5 minutes, and stored in -20C for western blot (2 days after the extraction). Ib the day of the western blot, I reboiled the sample again under the same condition, Then, I proceeded to the the western blot step.

7. The western blot is standard protocol, with the first antibody blocking overnight (~16 hours), and secondary antibody for 1 hour.

-------------------------------------

D. Result & My Analysis

The Problem is I got a band of 200 kDa even in the negative control of no hypoxia. And the result is consistent, I got his in 4 gels. However, I found that using the Laemmli buffer resulted in a clearer band in the 200 kDa region. I checked in the lower region of < 50 kDa there is some faint (pseudo) band, is the HIF1-A degraded?

I don't think it's a Western blot problem, since I consistently get the band of a housekeeping gene (beta-actin).

My main concern is the culture condition or my extraction protocol.

Or maybe the IEC-6 doesn't show any strong signal of HIF-1A in hypoxia conditions. Most papers used the cancer cells or endothelial cells for checking the HIF-1A.

Please feel free to add any suggestions,

Thank you in advanced