Anti-Cdc25A antibody [DCS-120 + DCS-121] (ab2357)

Customer kindly called to inquire about Ab2357 in IHC-P as they were getting reduced signal and higher background with the newest lot.

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1070441.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

what is the concentration?

ANSWER:

The concentration is 100 ug/ml.

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

ANTIBODY CODE ab2357 BATCH NUMBER 40683

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Cell lysates from human tumour cell lines

PRIMARY ANTIBODY CDC25a (DCS120 + DCS121) 1:1000 and 1:500 in milk left in frdge overnight, wash for 10 mins before putting in secondary

SECONDARY ANTIBODY anti mouse from DAKO 1:1000 incubate for 1hour wash for 1hour

DETECTION METHOD ECL from AMersham 5mls of each reagant, leave for approx 1 minute

POSITIVE AND NEGATIVE CONTROLS USED none as have none avaliable to me.

ANTIBODY STORAGE CONDITIONS 4 degrees C

SAMPLE PREPARATION lysate + laemmli buffer to 30ul. Heated to 94 degress for 10minutes before loading

AMOUNT OF PROTEIN LOADED up to 50ug

ELECTROPHORESIS/GEL CONDITIONS 4-20% gel (invitrogen)

TRANSFER AND BLOCKING CONDITIONS transfer for 1.5 hours at 160mAmps Blocking for at least 1 hour in 5% milk + PBS

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? dilution of Primary antibody

ADDITIONAL NOTES I have used the same method, the same secondary and the same ECL on other westersn which have worked. This antibody has also failed to work on IHC of paraffin embedded sections at a dilution of 1:100

ANSWER:

Thank you for your enquiry and providing more details of your protocol. This antibody has been optimized for paraffin tissue sections. We would suggest using at 1:25, 1:50 for 30 min. Pretreatment with Citrate buffer. Use tonsil as positive control along with a standard ABC kit (signal enhancing step). We think you are diluting antibody too much. Start with lower dilution and then move up with higher dilutions. If you need anything further or any help then please let us know.