Anti-DPP9 antibody - Aminoterminal end (ab42078)

Our customer has responded, please see below:

“If possible we would like the Catalytic region of DP9 ab which is ab42080 This has been used a lot in the literature and appears to work well in Westerns.”

ANSWER:

Thank you for kindly confirming these details as this enables us to closely monitor the quality of our products
We would be pleased to arrange a replacement product free of charge. I am forwarding these details on to our Distributor’s Team who would be happy to organize this.
Distributor name: ********
Original order number: ********
abID of original product: 1 X vial ab42078
abID of replacement product: ab42080
CCEID: ********
Thank you for your help and cooperation. Please do not hesitate to contact us if you need anything further.

LOT NUMBER 275780
ORDER NUMBER
DESCRIPTION OF THE PROBLEM Multiple bands
SAMPLE Wildtype SKOV3 and 293 human cell lines. 293T cells transfected with WT and mutant eGFP-fused DP8 and DP9 and peGFPN1.
PRIMARY ANTIBODY The aliquoted rabbit polyclonal anti –DP9 antibody was diluted 1:1000 blocking buffer (see above). Primary antibody incubation was overnight at 4ºC Membrane was washed with PBS with 0.1% Tween-20, 3 x for 10 min each
DETECTION METHOD 1 ml SuperSignal® West Pico Chemiluminescent Substrate (Thermo scientific) for 5 minutes in ambient lighting.
POSITIVE AND NEGATIVE CONTROLS USED Recombinant DP8 and DP9 and cell lines expressing peGFPN1
ANTIBODY STORAGE CONDITIONS -20ºC and made in 5µl aliquots
SAMPLE PREPARATION 1 x 107 cells were lysed in 300µl cell lysis buffer using a probe sonicator 2 x 10 second on ice Cell Lysis Buffer 150 mM NaCl 1 mM EDTA 1x Protease Inhibitor Cocktail (Sigma) In PBS pH 7.4 Cells were then spun in the ultra-centrifuge at 39000 rpm for 30 minutes at 40C The resulting supernatant was removed and protein concentration then determined Enough lysate to give 50 (figure 3) or 100 µg of protein was then mixed 2:1 with 3 x SDS-PAGE loading buffer 3 x SDS-PAGE loading buffer 3% w/v SDS 93.75 mM Tris-HCl pH 6.8 0.015% w/v Bromophenol blue 37.5% v/v Glycerol 7.5% v/v 2-Mercaptoethanol Samples were heated for 5 min at 100°C After heating the sample was loaded onto a precast 10 lane SDS-PAGE
AMOUNT OF PROTEIN LOADED 50 (figure 3) and 100 µg cell lysates, 120 ng recombinant DP8 and 240 ng recombinant DP9
ELECTROPHORESIS/GEL CONDITIONS 10% and 4-20% Mini-PROTEAN® TGX™ Precast gels (BioRad) and run in running buffer (0.025M Tris, 0.192M Glycine and 0.1% SDS) at 170 V for 70 minutes.
TRANSFER AND BLOCKING CONDITIONS Transfer was conducted in 1 x transfer buffer at 60V for 90 min 1 x Transfer buffer 0.025M Tris 0.192 M Glycine 10% Methanol 0.025% SDS Membrane was blocked by incubating in blocking buffer for one hour at room temperature with rocking Blocking buffer 3% skim milk powder PBS pH 7.4 0.5M NaCl 0.1% Tween-20)
SECONDARY ANTIBODY Swine anti rabbit-HRP from Dako, diluted 1:2500 in blocking buffer Secondary antibody was incubated for 1 hour at room temperature Membrane was washed 3 x 10 min each with PBS + 0.1% Tween-20
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10 ti
HAVE YOU RUN A "NO PRIMARY" CONTROL? No
DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? Loaded 48 ng recombinant DP8 and 96ng recombinant DP9
ADDITIONAL NOTES PROBLEM:
The anti-DP9 Ab cross reacts with DP8. This antibody was ordered and shipped on the 28th June 2011 (Invoice No S123911, 28/6/11). Previously in our lab we have been using the same Ab sourced from Triple Point Biologics and we have not experienced this cross-reaction problem.
The first time this Ab was used was 13th September 2011 (see Fig 1) and at this point it cross-reacted as described below Figure 1. Transient transfection of 293T cells with peGFPN1, wild type eGFP-fused DP8 and wild type eGFP-fused DP9. 50 µg protein of cell lysate was loaded into 10% precast gel. In Figure 1, the anti- DP9 ab does not detect recombinant GFP-fused DP8 in the lysates but it does bind to recombinant DP8 ( Lane 6). Figure 2. Transient transfection of 293T cells with peGFPN1, wild type eGFP-fused DP8 and eGFP-fused DP9 and mutant eGFP-fused DP8 and eGFP-fused DP9. 50 µg protein of cell lysate was loaded into 10% precast gel on 31st October 2011. In Figure 2, the DP9 Ab is cross reacting strongly with DP8-GFP wt ( Lane 1) and weakly with DP8-S739A(Lane 2) mutant forms.
It reacts very strongly with the DP9 and DP9 wt GFP fusion proteins (Lane 3, 5 and 6) and it detect endogenous DP9 in the cells (Lane 7 and 8) and detects recombinant purified DP9. Figure 3. DP9 ab42078 against 120 ng DP8 and 240 ng DP9 recombinant protein and lysates of THP-1 (50 μg protein) and Caco-2 cells (20 ug protein) In this image the DP9 Ab is reacting with recombinant DP8 that has been loaded as a control.
In summary the anti-DP9 ab does not seems to detect endogenous DP8 but it is detecting recombinant purified DP8 and also over-expressed DP8 fused to GFP- intermittently. This is probably dependent on the transfection efficiency so if lot of fusion protein present it binds to it.

ANSWER:

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.
Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.
Unfortunately we currently have in stock the lot 275780.
I apologize for the inconvenience and am pleased to offer you in compensation a credit note or a free of charge replacement with an alternative anti-DPP9 from the list below (excluding ab42078 of course) :
http://www.abcam.com/index.html?pageconfig=searchresults&search=dpp9&pt=1&sk=react&sv=21&sn=Human&l=2&fViewMore=1
Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.