Anti-COX2 / Cyclooxygenase 2 antibody [SP21] (ab16701)

BATCH NUMBER 237901 ORDER NUMBER 196732

DESCRIPTION OF THE PROBLEM There are strong bands at the wrong band sizes (see attached file).

SAMPLE Rat liver hepatoma, H4IIE-cells treated with TNF-alpha

PRIMARY ANTIBODY abcam, ab16701-500 Rabbit monoclonal [SP21] to COX2/ Cyclooxygenase 2 diluted 1/200 in 1% milk powder in TBS-Tween incubation time: 2.5 hours at room temperature agitating wash step: 3 x 5 min with TBS-Tween

DETECTION METHOD ECL, substrate and peroxide buffer from tebu-bio : Chemiluminescent Sensitive HRP Substrate Kit (SuperDetect);2-Component Chemiluminescent Substrate for the Detection of Horseradish Peroxidase. Detection with Lumi-Imager from Roche (Lumi-Analyst Software) or X-ray films.

ANTIBODY STORAGE CONDITIONS +4°C in storage buffer (data sheet)

SAMPLE PREPARATION H4IIE-cells were treated with different concentrations of TNF-alpha for 6 hours. Incubation scheme: 1 000 000 cells per 60 mm petri dish were sowed in DMEM low glucose containing 10% BSA and 1% P/S and left to adhere for 24 hours at 37 °C. Then the culture medium was evacuated and the cells were washed with 0,9 % sodium chloride. Then The cells were incubated with 10, 30, 50 or 70 ng/ml TNF-alpha (dissolved in culture medium) for 6 hours. After 6 hours the incubation medium was evacuated and the cells were washed three times with 0,9 % sodium chloride. Then the cells were incubated with culture medium for another 24 hours. After 24 hours the medium was evacuated and cells were washed with 0,9 % sodium chloride. Cell lysis has been carried out by harvesting the cells with 70 µl RIPA-Lysis-buffer per 60 mm dish (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1% Triton X-100, 1 % sodium deoxycholate, 0,1 % SDS, 50 µl Protease Inhibitor Cocktail from sigma). Cell suspension was then incubated for 15 min on ice with gentle agitation and centrifugated for 15 min with 14 000 x g at 4 °C. Supernatant was evacuated and stored at -80°C until protein determination by Bradford Assay.Samples were diluted with RIPA-buffer to 4 µg/µl total protein. From this solution 12 µl were diluted with 6 µl Laemmli buffer (3x) and heated up to 95 °C for 5-6 min. The samples were pulsed and loaded onto the gel.

AMOUNT OF PROTEIN LOADED 48 µg total protein per lane, protein determined by Bradford Assay

ELECTROPHORESIS/GEL CONDITIONS stacking: 4 % SDS-polyacrylamidgel separating/resolving: 10% SDS-polyacrylamidgel

buffer: 2 M glycine, 250 mM Tris, 1 % SDS run: 70 V for 15 min, 100 V for 1 h 40 min

TRANSFER AND BLOCKING CONDITIONS discontinuous buffer system: anode buffer I : 300 mM Tris, 10 % methanol, pH 10.4 anode buffer II: 25 mM Tris, 10 % methanol, pH 10.4 cathode buffer: 40 mM glycine, 25 mM Tris, 10 % methanol, SDS, pH 9.4

time period: 1 h 25 min at 90 mA per membrane PVDF membrane (Millipore)

blocking conditions: 1 h at room temperature with 5 % milk powder in TBS wash step after blocking: 3 x 5 min with TBS-Tween

SECONDARY ANTIBODY [another company]./ goat anti rabbit HRP-linked diluted 1/1000 in 1 % milk powder in TBS-Tween incubation time: 1.5 hours at room temperature agitating wash step: 3 x 5 min with TBS-Tween

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? washing steps reduced, electrophoresis run extended, blotting time extended, new dilution of frozen samples,

ADDITIONAL NOTES The attached file shows the last western blot. Loaded samples: H4IIE-Lysate (as described above) from left to right: 10, 30, 50, 70 ng/ml TNF-alpha incubation (6h); 48 µg total protein per lane. Two different times of exposition (30 sec and 300 sec) . Detected with chemiluminescence substrate from tebu-bio and Lumi-Imager (Roche).

ANSWER:

I'm very sorry to hear you are experiencing problems with ab16701.

The protocol you have used is very good and should work well; in light of your problems it is clear that the antibody did not work well in western blotting.

I would like to offer you a refund or credit note on your order, or another anti COX2 antibody of the same value as ab16701. Could you please tell me which you would prefer and I will arrange this immediately,

I look forward to hearing from you to arrange this,

The catalog number I meant to inquire about is ab16701.

ANSWER:

Thank you for your enquiry.

I am not sure that I follow your question, please can you elaborate.

ab16701, anti-COX2 recognizes both the rat COX2 (69164KDa) and the human COX2 (68996KDa).

I hope this information helps. Please do not hesitate to contact me should you require further assistance.

For WB with ab16701, what type of lystae is a good positive control?

ANSWER:

Our source for this antibody has recommended HT 29 lysate as a positive control for Western blotting.

This type of lysate can be obtained from Abcam; it is catalog # ab3952.

If you have any additional questions, please contact us again.