Application
Western blot
Sample
Human Cell lysate - whole cell (primary cancer-associated fibroblasts (prostate))
Loading amount
30 µg
Specification
primary cancer-associated fibroblasts (prostate)
Gel Running Conditions
Reduced Denaturing (Invitrogen's NuPAGE 4-12%)
Blocking step
BSA as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Other product details
Dilution
1/2000
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: PBS + 0.075% Tween20 + 5% BSA
Secondary antibody
Name
Non-Abcam antibody was used: Goat-anti-mouse IgG (H+L), Invitrogen & Molecular
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 680
Host species: Goat
Clonality: Polyclonal
Conjugation: Alexa Fluor® 680
Dilution
1/10000
Detection
Detection method
fluorescence via Licor Odyssey
Exposure
4 minute(s) and 0 second(s)
Bands
Specific: ~90 kDa
Positive control
my sample
Additional data
Additional Notes
This is from my optimisation experiment:
I used 30 ug cell lysate per lane and the AB dilutions were 1/2000 (lane 1) and 1/4000 (lane 2). I recommend to use it at 1/2000 dilution and with BSA as blocking agent instead of milk powder, as signals were much weaker with milk powder blocking (bright red spots derive from air bubble).
Marker: Invitrogen's SeeBluePlus2, 7ul per lane.
Scanning intensity on Licor Odyssey: 5.0.
I used 30 ug cell lysate per lane and the AB dilutions were 1/2000 (lane 1) and 1/4000 (lane 2). I recommend to use it at 1/2000 dilution and with BSA as blocking agent instead of milk powder, as signals were much weaker with milk powder blocking (bright red spots derive from air bubble).
Marker: Invitrogen's SeeBluePlus2, 7ul per lane.
Scanning intensity on Licor Odyssey: 5.0.
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.
Dr. Alexander Henke
Verified customer
提交于 Aug 02 2011