Western blot abreview for Anti-Carbonic Anhydrase IX antibody

Western blot

Human Cell lysate - whole cell (MCF-7, 786-O, RCC4)

20 µg

MCF-7, 786-O, RCC4

MCF-7: 21% oxygen vs 1% oxygen for 20 hours

Reduced Denaturing (Pierce precise protein gel 4-20%)

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

1 µg/ml

16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% Milk, PBS-Tween 0.1%

Non-Abcam antibody was used: swine anti-rabbit HRP
Host species: Pig
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase

1/5000

supersignal west dura

10 minute(s) and 0 second(s)

Specific: 65 kDa Non-specific: 47 kDa

RCC4 lysate (PMID: 15964822)
MCF7 lysate (PMID: 16847340)

786-O lysate (PMID: 15964822)

Lane order 1-4: MCF7 Normoxia, MCF7 Hypoxia, 786-O, RCC4
This antibody detects a species of ~65KDa by SDS-PAGE which is slightly higher than the predicted Mw of CA9 (50 kDa). However, both positive and negative controls strongly suggest that this 65 kDa species is CA9.
CA9 expression is regulated by hypoxia and, more specifically, by the HIF-1a isoform (see PMID: 15964822). Thus, CA9 is highly expressed in VHL-defective RCC4 cells which express HIF-1a but not in the related VHL-defective cell line 786-O which lack HIF-1a. Finally, a hypoxia inducible species of the same mobility is observed in MCF7 lysate.