Western blot abreview for Anti-PAX8 antibody

Below Average
Abreviews
Application
Western blot
Sample
Xenopus tropicalis Cell lysate - whole cell (Whole embryo)
Loading amount
20 µg
Specification
Whole embryo
Gel Running Conditions
Reduced Denaturing (4-12%)
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Other product details

Dilution
1/3000
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5%MILK or 3%BSA

Secondary antibody

Name
Non-Abcam antibody was used: Jackson HRP goat anti-rabbit
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/20000

Detection

Detection method
ECL
Exposure
2 minute(s) and 0 second(s)
Bands
Specific: 50 kDa Non-specific: 195, 100, 40, 45, 43, 28 kDa
Positive control
I do not have a positive control.
Negative control
The third lane in the WB is a mutant extract that DOEs Not have the pax8 protein. Still the pattern of bands is the same than for lane 2 (WT extract). The 50 KDA band that could be the specific one is present also in the mutant so it is not Pax8.

Additional data

Additional Notes
Pax8 protein is around 50 KDa (in X. tropicalis), there is a band of around that weight, but is present in the 3 lanes. In the MUT lane (3) it should be absent. Besides We needed a more specific antibody (potentially to used it for ChIP) and this one is detecting too many unspecific bands.
For WB I have done lower dilutions (1/1500 and 1/1000) that gave me more unspecific bands.
I have also tried the antibody ab97477 for IHC but didn't work. (dilution 1/100 and 1/200)
Abcam response
Thank you for your review. This is the first data we have for this species and we expect cross-reaction, given that the immunogen shares 93% identity with the X. tropicalis PAX8. If the mutation is a functional mutation, there is a possibility that the antibody is still capable of recognizing the non-functional PAX8, but we agree that there is too much cross-reaction with other elements in the lysates to recommend this antibody for ChIP with your samples.

Dr. Florencia del Viso

Verified customer

Submitted May 20 2011

Sign up