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The antibody ab8637 in the following order doesn't work well. The detailed informed is included in the attached file. Could you please give us some help?
Asked on Aug 20 2012
Thank you for contacting Abcam regarding ab8637.
I am sorry that you are experiencing difficulties with this antibody in WB. In order to assist you further, I wanted to clarify several points in your protocol.
You mention that you load 15ul of protein - do you know what the quantity of protein is in ug? Was the protein isolated from a particular cell type (tranfected)or translated in vitro (purified protein)? What is the difference between each of the three samples?
Here are the recommended WB protocol conditions:
Following lysis, prepare samples in Laemmli buffer and boil 10 minutes prior to loading.
Load 50ng of purified protein or 50 - 100ug cell lysates
Prior to WB, confirm efficient transfer to the membranes by Ponceau S
Block - 5% milk (or preferrably BSA) in TBSTfor 1 hr at RT
Primary antibody - dilute ab8637 1:1000 - 1:5000 in 5% milk (or BSA) and incubate o/n at 4oC or 1 hr at room temperature
Wash 3X 10 min in TBST
Secondary antibody (HRP conjugated)- dilute according to manufacturers instructions and incubate 1 hr at RT
Wash 1X 15 min and 3X 10 min in TBST
Develop using ECL
Based on the results you provided, you are getting the band of interest with some background that can be cleaned up by modifying the protocol a bit. I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions.
Answered on Aug 20 2012