Anti-Amyloid Precursor Protein antibody [DE2B4] (ab12266)

I want to use the Abcam anti-APP (DE2B4) antibody #ab12266 in a collaboration to test a newly developed technique. The collaborators now ask me if I can provide affinity of the antibody for the abeta peptide. As I didn't find this information on the product sheet, I wondered if you measure affinity during development or production of the antibody.

ANSWER:

Thank you for contacting us. A collaborator has used ellipsometry to measure the affinity of this antibody for a synthetic peptide corresponding to amino acids 1-16 of beta peptide and gets a value of around 10-8 M.

This is a very preliminary measurement, but it may provide a starting point for your technique. I hope this helps, please let me know if you need any additional information.

The researcher would like to know the fixative used in ICC with ab12266.

ANSWER:

I received the following information from the researcher who tested the antibody ab12266 in ICC: "We routinely use ice-cold methanol fixation for 10 minutes, followed by a brief wash with TBS. We have also used 4% paraformaldehyde for 20 minutes followed by permeabilization with either methanol of 0.5% Triton X-100 in TBS and this also seems to work fine."

Please let me know if you need further assistance,

BATCH NUMBER 176737 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE I have transfected the DNAs encoding either APP or factor A into human cancer cells. The number of cell was counted and resuspended in appropriate amount of 2X SDS-PAGE sample buffer and boiled for 3min.

PRIMARY ANTIBODY The membrane was blocked with 5% skim milk in TBS-T buffer. Anti-APP antibody was diluted at first 1:500 and later 1:100 and added to the membrane and incubated overnight with gentle rocking. The membrane was incubated further 30min at RT, washed three times with TBS-T

DETECTION METHOD After 3 times washing (10min for each washing), ECL solution (Amersham-Pharmacia) was added, incubated for 1 min and the membrane was developed with Hyperfilm (Amersham-Pharmacia) by exposing the film for 5 min, 20 min, 2 hours, or overnight

POSITIVE AND NEGATIVE CONTROLS USED I also tried to detect endogenous APP in the same cell line by using another company's antibody and there was no problem for detection.

ANTIBODY STORAGE CONDITIONS 4C

ELECTROPHORESIS/GEL CONDITIONS 8% SDS-PAGE

TRANSFER AND BLOCKING CONDITIONS transferred activated PVDF membrane for 90 min at 60V

SECONDARY ANTIBODY secondary antibody (anti-mouse) was added and incubated for 2 hours.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? See above

ADDITIONAL NOTES While i was filling the complaint report, i repeated the experiment with fresh primary and secondary antibody and i still could not get the target band. There were several faint background bands but no APP specific band.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that your customer has been having difficulties using this antibody by western blotting. Their approach is largely on that I would recommend. They have tried various dilutions using an overnight incubation. I am certainly prepared to offer your customer credit if the antibody was purchased within the past 90 days. If this is the case please e-mail me details of the order including the date of purchase and order number.

How does your new anti-beta amyloid 1-17 differ from 6E10? Do any of your antibodies work for ELISA for beta amyloid in human serum/plasma? Finally, what is titer per mg mAb protein? Affinity?

ANSWER:

Thank you for your enquiry and your interest in our products.

Ab12266 and ab10146 are from two different clones and sources. They have been tested for different applications.

For further information, please take a look at the on-line datasheets and decide which one would suit your need best.