Application
Western blot
Sample
Human Cell lysate - whole cell (HUVEC and HAEC)
Loading amount
25 µg
Specification
HUVEC and HAEC
Treatment
with or without VEGF
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Other product details
Dilution
1/500
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% BSA TBS/T
Secondary antibody
Name
Non-Abcam antibody was used: Goat anti-rabbit HRP
Host species: Goat
Clonality: Monoclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Monoclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/2500
Detection
Detection method
ECL+
Exposure
30 minute(s) and 0 second(s)
Additional data
Additional Notes
The antibody apparently doesn't have high affinity to NP1. Both HUVEC and HAECs were tried (both reportedly has quite a few NP-1 present) and both didn't show any band. This is a disappointment. I will try include a positive control with cells overexpressed with NP-1. But basically, if it can't even detect NP1 in normal human endothelial cells, I see little value in such antibody, at least definitely not for western.
Abcam response
I'm sorry you are experiencing difficulties with this antibody. To our knowledge, this antibody has not yet been tested on endogenous protein. I would recommend increasing the primary antibody concentration. Increasing the detergent strength may also improve results, as this is membrane-associated protein.
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.
Abcam user community
Verified customer
投稿 Oct 31 2007