Application
Western blot
Sample
Pig Cell lysate - whole cell (kidney, LLCPK1-F+)
Loading amount
20 µg
Specification
kidney, LLCPK1-F+
Treatment
Cells grown in pH 7.4 or pH 6.9 media
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
(agent) for 2 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Other product details
Dilution
1/1000
Incubation time
20 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: Blocking Buffer with 0.1% Tween 20
Secondary antibody
Name
Non-Abcam antibody was used: Licor IRDye 800 CW Goat Anti-Rabbit IgG
Host species: Goat
Clonality: Polyclonal
Conjugation: IRDye® 800CW
Host species: Goat
Clonality: Polyclonal
Conjugation: IRDye® 800CW
Dilution
1/10000
Detection
Detection method
Licor Odyssey
Exposure
1 second(s)
Positive control
None.
Negative control
None.
Additional data
Additional Notes
20 ug of LLCPK1-F+ pig kidney cell lysate loaded per lane. Lane 1, pH 7.4. Lane 2 , pH 6.9. This cell line does not readily express p37 AUF1. Previous overexpression of p37 AUF1 showed that it migrates slightly faster than p40/42 AUF1, and never faster than the 37 Kd marker. The anti-phospho (S83) antibody should only recognize the p40 and p45 isoforms: S83 is located in exon 2, which is not present in the p37 and p42 isoforms. This antibody did not recognize the p40 or p45 isoforms. Immobilon-FL membrane used for transfer. RIPA lysis buffer with HALT phosphatase inhibitors added.
Abcam response
Thank you for your valuable feedback. We are sorry to hear that this antibody has not given you good results in western blot using pig sample. This antibody has not been tested in this application nor species before, and it is possible that it may not suitable for use.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Ms. Lakshmi Gummadi
Verified customer
投稿 Jan 30 2012