You have changed your country from to
. Please be aware that this will change the currency in the purchasing process.
Related products
- Secondary antibodies
Reassurance, Refunds & Replacements
If your product does not perform as described on this datasheet, we will refund or replace your product...
Read our guarantee »This product is covered by the Abpromise guarantee. Our scientific support team are available to answer any questions or queries - fill out an inquiry form for ab14223 for help.
Alternatively, you can search the previous enquiries about this product to see if your query has already been answered.
|
|||||||||
 | |||||||||
|
|||||||||
BATCH NUMBER 164531 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE 3T3L1 cells (mouse) induced with TNF alpha and also uninduced cells they know message is going up by real time (measures amount of mRNA) whole cell extracts with protease inhibitors PRIMARY ANTIBODY 1:400 and 1:500 diluted in TBS-T for overnight at 4C standard 3X wash in TBS-T DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED 3T3L1 cells (mouse) induced with TNF alpha and also uninduced cells they know message is going up by real time (measures amount of mRNA) ANTIBODY STORAGE CONDITIONS 4C for short term SAMPLE PREPARATION protease inhibitors sample heated at 70 for 10 minutes with reducing agents ponceau is positive AMOUNT OF PROTEIN LOADED 20 ug ELECTROPHORESIS/GEL CONDITIONS reducing 10% gel TRANSFER AND BLOCKING CONDITIONS Novex (invitrogen) standard transfer buffer 1 hr at 130V Using Rainbow MW ladder Transfer also checked by ponceau staining 5% NFDM in TBS-T SECONDARY ANTIBODY ab6877 (goat anti-chicken HRP) (lot 151196) 1:500 (only dilution that produced signal and that was below wells - still weak), 1:1,000, 1:5000, 1:7,500 diluted in TBS-T 3hr at RT Standard 3X wash w/ TBS-T HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4+ HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Primary dilutions and secondary dilutions ADDITIONAL NOTES Upregulation of protein confirmed by RealTime PCR Transfer confirmed by Ponceau
|
|||||||||
ANSWER: |
Thank you for your enquiry. I am sorry to hear that your customer has been having difficulties with this antibody. To our knowledge this antibody has only been tested on an E coli-derived fusion protein as the test antigen. I am not sure where the recommended dilution of 1:500 was derived from and have therefore removed it from the datasheet. I apprecaite that your customer has been detecting an upregulation of TNFAIP3 by real time RT-PCR analysis. However, to improve the detection of the TNFAIP3 protein I would like to recommend that you customer increases the mass of protein that they are loading on the gel to ~50ug, decreases the dilution of the antibody to ~1:200 and applys BSA rather than milk as a blocking agent. WE frequently find that BSA produces cleaner, more sensitive and specific blots. I think that this will enable your customer to fully determine whether it is the antibody that is compromised. |
||||||||
|
|||||||||
What is the protein concentraton for lot 160353? |
|||||||||
ANSWER: |
Thank you for your patience. The concentration is 1 mg/mL. |
||||||||
|
|||||||||
| |||||||||
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
