Anti-PARK7/DJ1 antibody (ab4150)

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

DESCRIPTION OF THE PROBLEM Non-specific bands with mouse liver extract.

SAMPLE mouse liver extract

PRIMARY ANTIBODY AB4150

SECONDARY ANTIBODY [another company] rabbit anti goat

DETECTION METHOD radiolabeled

ANTIBODY STORAGE CONDITIONS aliquots frozen at -20?C then stored at 4?C after thawing

AMOUNT OF PROTEIN LOADED 10?g extract

ELECTROPHORESIS/GEL CONDITIONS reducing

TRANSFER AND BLOCKING CONDITIONS Towbin, 2hrs

ADDITIONAL NOTES Have you tried this antibody with mouse ? In my hands, it generates a lot of non-specific bands. Thoughts appreciated.

ANSWER:

Thank you for your enquiry. Yes, this antibody has been tested for cross-reactivity with both mouse and human. On the online datasheet for ab4150 there are two Western blot images, the first one using Jurkat lysate and the second one showing HeLa whole cell lysate and 3T3 whole cell lysate.

Have you tried a positive control? We recommend Jurkat lysate. You didn't mention what dilutions you have tried, but to try to reduce the number of non-specific bands, I suggest the following: Decrease the amount of primary antibody that you are using as well as decrease the incubation period time. Decrease the amount of secondary antibody that you are using and also make sure to run a secondary antibody control (no primary antibody) to ensure these bands are not due to your secondary antibody.

Please let me know if you need further assistance.

BATCH NUMBER 43997 ORDER NUMBER z759557

DESCRIPTION OF THE PROBLEM No signal

SAMPLE M17 human neuroblastoma cell protein extract, whole mouse brain extract

PRIMARY ANTIBODY 1:1000 in PBS+5%milk overnight. Wash 3X 10 minutes in PBS-Tween20

SECONDARY ANTIBODY anti-rb goat HRP, 1 hour at room temperature, 1:2000.

DETECTION METHOD ECL plus

ANTIBODY STORAGE CONDITIONS -20

SAMPLE PREPARATION Sigma protein lysis buffer

AMOUNT OF PROTEIN LOADED 10ug

ELECTROPHORESIS/GEL CONDITIONS 4-20%

TRANSFER AND BLOCKING CONDITIONS Transfer buffer, 4degrees overnight at 30volts, block with 5%milk

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Tried different samples.

ANSWER:

I'm sorry to hear you are having a problem with our PARK7 (DJ1) antibody ab4150.

I would like to suggest the following modifications to your protocol: - The protein subcellular localisation is described as both nuclear and cytoplasmic, I would therefore suggest using nuclear and cytoplasmic extracts of your samples rather than whole cell extracts.

-Load more protein per well (40-50ug) and use more concentrated primary antibody (try 1:250, 1:500) (still overnight at 4C)

-check the transfer is working properly by doing a Ponceau red stain

-use a positive control such as Jurkat cell lysate.

I hope these suggestions will help, do not hesitate to contact us for further advice,

DESCRIPTION OF THE PROBLEM I tested this antibody on adult mouse brain lysate(50ug per lane). I got one single 28kd band(I cut the membrane, only use the part lower than 35kd). The protein marker was from Bio-Rad prestained SDS-PAGE standards low range 161-0305

SAMPLE adult mouse brain whole protein lysate(50ug per lane)

PRIMARY ANTIBODY 1:1000 in 6% milk, 4hour R.T.

DETECTION METHOD supersignal pico

POSITIVE AND NEGATIVE CONTROLS USED no

ANTIBODY STORAGE CONDITIONS 4deg for only 3 days upon receiving.

AMOUNT OF PROTEIN LOADED 50ug

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

Thank you for your enquiry and patience. It is interesting that you got a single band at 28 kDa. The predicted molecular weight of Park7/DJ-1 is 19.8 kDa (Swissprot Accession Number Q99497) and an approximately 20 kDa band was seen in Jurkat lysate when this antibody was tested.

However, in the literature there is wide variation in reported molecular weights for this antibody and two other distributors have reported that this clone detects a band at 28 kDa.

We are currently looking into this issue further. Our antibody has not been tested before with mouse samples, only human, so we unfortunately cannot give any guarantees regarding how the antibody works with untested species. My recommendation is to run a positive control - Jurkat lysate.

If you have any more questions or comments, please do not hesitate to contact us again.

Customer says that other monoclonals to PARK7 recognize a 28 kDa band, so why does ab4150 recoginze a 20 kDa band?

ANSWER:

Thank you for your enquiry. You raise an interesting point and we have taken it very seriously. As you may know the predicted molecular weight of Park7/DJ-1 is 19.8 kDa (Swissprot Accession Number Q99497) so a band of 28 kDa is substantially larger than predicted. Furthermore a quick survey of the literature on this antibody shows wide variation in reported molecular weights:

- As you say, the two distributors claim 28kDa - Le Naour et al in Clin Cancer Res. 2001 Nov;7(11):3328-35 use the same antibody and find reactive spots at around 25kDa using 2D-PAGE - Bandopadhyay et al in Brain. 2004 Feb;127(Pt 2):420-30 again use the same antibody in 1D and 2D PAGE and clearly show it running at 20KDa!

So our result does not actually seem inconsistent with the literature and the predicted size of the protein.