Anti-PPAR alpha antibody (ab24509)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM 2 bands one at 50kd and 52 kd

SAMPLE mouse/nuclear

PRIMARY ANTIBODY ABcam PPARa/ Rabbit/ 5ug/ml/ incubate overnight, wash 3xTBST.

DETECTION METHOD Amersham/GE Health ECL solution

POSITIVE AND NEGATIVE CONTROLS USED k562 whole cell lysate used 20ug

ANTIBODY STORAGE CONDITIONS -20 freezer

SAMPLE PREPARATION Invitrogen nupage kit. Heating samples for 10 min at 70c

AMOUNT OF PROTEIN LOADED 25 ug / lane

ELECTROPHORESIS/GEL CONDITIONS Reducing, 4-12% Bis tris pre-cast gel. Mops-SDS running buffer. Running gel for 50 min at 200V.

TRANSFER AND BLOCKING CONDITIONS Block for 2 hours in 5% milkin in TBST. Wash 3X with TBST.

SECONDARY ANTIBODY [another company]/Rabbit/1:2500/2hr/wash 3x with TBST

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No

ADDITIONAL NOTES Will rat show a band at 52Kd. or would I expect to not see a band?

ANSWER:

Thank you for your enquiry. After contacting the originator of this antibody, I have confirmed that this antibody does in fact react with rat, and that change will be made to the datasheet. I would expect you to get the predicted band if you used rat samples.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Thank you for your reply. Please refer to the customer's response:

I only saw that 50 kD band in the rat liver lysate not rat muscle lysate. I am trying to measure PPAR alpha in rat muscle. That is my problem your antibody is not picking up PPAR alpha in rat muscle.

Would you have any other comments/suggestions?

Kind regards

ANSWER:

Thank you for getting back to me.

From literature searching I can see that the detection of PPAR alpha in rat muscle is to be expected. Given that your customer has been preparing their samples in the same manner and has been detecting the non specific band at ~60 kDa in all of their samples I find it concerning that they have been unable to detect a 50KDa band in their skeletal muscle samples.

After consideration I am prepared to offer your customer credit against this purchase provided that they purchased the antibody within the past 90 days. If this is the case please email me details of the initial order including the order number and date of purchase.

BATCH NUMBER 176668 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Non-specific band. In rat muscle lysate I am getting two bands around 50 and 60 kD, one of them is right at 60 kD and one is around 56-58kD but nothing at 52 kD or around 50 kD. In rat liver lysate I am getting 3 bands around 50 and 60 kD, one is right at 60 kD, one is at 56-59 kD and one is right at 50 kD

SAMPLE Rat muscle lysate

PRIMARY ANTIBODY Abcam PPAR alpha, Rabbit polyclonal, 1:1000 overnight 4C

DETECTION METHOD West Femto Supersignal

POSITIVE AND NEGATIVE CONTROLS USED Rat Liver Lysate

ANTIBODY STORAGE CONDITIONS -20C undiluted, diluted stored at 4C

SAMPLE PREPARATION All samples are homogenized in the same buffer all protease and phosphotase inhibitors used, samples are aliquoted, boiled 5 mins and stored in -20C

AMOUNT OF PROTEIN LOADED 60 micrograms

ELECTROPHORESIS/GEL CONDITIONS SDS ?PAGE, 10% gel, 160V for 1 hr

TRANSFER AND BLOCKING CONDITIONS Semi-dry transfer, 1hr at 10V, blocked overnight at 4C in 5% NFDM

SECONDARY ANTIBODY Amersham Bioscience, anti-Rabbit, 1:2000, 1 ? hours

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10+ HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Incubation on primary/secondary, samples

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionaire and I have a few comments. On the antibody datasheet it details that when using a human erythroid cell line (K562) three bands are detected: 52 kDa and ~60 kDa band of similar densities and a much weaker band at ~45-50 kDa. Given your customers observations I consider it very likely that the three bands that they have been detecting in rat cells are the three bands mentioned. The minor deviation in molecular weight (50KDa as opposed to 52KDa) is most likely attributable to different molecular weight standards employed in each case. It is well known that commercially available molecular weight standards migrate faster than others.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.