Anti-PINK1 antibody (ab23707)

BATCH NUMBER 198904 ORDER NUMBER 262791

DESCRIPTION OF THE PROBLEM At least three bands were detected from mouse/human cells and rat cortex tissues, and the major bands are 30-40kDa. Rat liver was also used as a possitive control, but the ~66kDa band was not detected.

SAMPLE mouse/rat MES cell extract, MES mitochondtria extract, human SH-SY5Y cell extract, rat cortex homogenate, rat liver homogenate and membrane fraction

PRIMARY ANTIBODY ab23707, 1:250 (4ug/ml) or 1:500 in 1%BSA/TBS-T, 4C O/N. Washes were 15'x2 and 10'x2.

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Rat liver was used as positive control, but 66kD band was not detected.

ANTIBODY STORAGE CONDITIONS stored in -20C as aliquots

SAMPLE PREPARATION For cell extracts, the lysis buffer was 25mM Tris (pH7.4), 150mM NaCl, 1mM EDTA, and 1% NP-40 or 0.5% Sarkosyl. Protease inhibitor cocktail from Sigma was also added. MES mitochondtria fraction was extracted using a PIERCE kit (89874).

Tissues were homogenated in 320mM sucrose, 5mM HEPES (pH7.4), 1mM EDTA and protease inhibitor cocktail. The rat liver membrane fraction was prepared as descibed in your Q&A section (Question 3):

1. Place tissue in lysis buffer (recipe below*) 2. Homogenize ~ 2 mins. 3. Centrifuge ~ 1300 x g for 20 mins. 4. Decant and reserve supernatant for later testing. 5. Resuspend cell pellet in desired buffer for use in SDS-PAGE. 6. Bradford to determine protein concentration 7. Dilute sample 1:1 with 2X SDS PAGE Buffer 8. Boil 2 mins. 9. Chill, then ready for Western Blot.

AMOUNT OF PROTEIN LOADED 10-50ug. 30ug as shown in the attached picture. Similar results were obtained using different amounts of total protein.

ELECTROPHORESIS/GEL CONDITIONS 8-16% SDS-PAGE, 160V ~1hr

TRANSFER AND BLOCKING CONDITIONS Transfer: 90V ~2hr, or ~0.3A O/N Blocking: 1-3hr in 5-10% milk in TBS-T buffer, R.T.

SECONDARY ANTIBODY Goat anti-rabbit (HRP) from Sigma, 1:20k in 1%BSA/TBS-T, 40-50min R.T. Washes were 5'x2, 10'x2 and 15'x4.

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

WHAT STEPS HAVE YOU ALTERED? blocking conditions, primary and secondary Ab concentrations

ADDITIONAL NOTES I was supposed to see 66kD and 33kD bands in rat liver, but I only saw the lower one. So how should I to improve the process to be able to see the 66kD band? Do you have a detail protocol? And for the cell extracts and the cortex tissue, do you have any suggestion to get rid of the bands of the wrong sizes? It looks that I have two 30-40 kD bands (not only the 33kD one) and they are the major bands.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties using this antibody. You are applying this antibody in a manner that I would recommend; preparing your rat liver lysate in the manner as we suggest and applying the antibody at the dilution that I would recommend.

Pink1 is a cleaved protein and I would like to suggest that the proteins that you are detecting are the result of cleavage. According to the literature (see below) PINK1 is cleaved and localises to the mitochondria. This may explain why you have observed only proteins within the region of 30-40KDa.

http://www.blackwell-synergy.com/doi/full/10.1111/j.1471-4159.2006.03845.x

Can you tell me whether you have evidence your samples contain the uncleaved, 66KDa product?

BATCH NUMBER 153790 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM I used this antibody before (lot number 143130) and i was getting really good specific results with using positive control rat liver. Now that i have bought in a new antibody (lot number 153790) the immunorecativity is decreased and that bands are coming up differently

SAMPLE The sample si am using are human brain material from tempoarl cortex

PRIMARY ANTIBODY tHE SPECIES IS RABBIT USED AT 1:500 MADE UP IN BLOCKING BUFFER. wASHES ARE ALWAYS 1*15MINS AND 3*5 MINUTES.

DETECTION METHOD Always ECL

POSITIVE AND NEGATIVE CONTROLS USED Poitive control used is rat liver

ANTIBODY STORAGE CONDITIONS Aliutoed on arrival and stored at -20oC

SAMPLE PREPARATION The buffer is PIPES-KOH, the protease inhibitors are PMSK,LEUPPETIN, APROTININ AND PEPSTATIN, The sample sare boiled for mins in lamelli buffer

AMOUNT OF PROTEIN LOADED We are loading 15ugprotein/25ul

ELECTROPHORESIS/GEL CONDITIONS It is reducing conditions (6%, BME) the gels are 10%, and they are ran at 120vlots for approx. 3Hours

TRANSFER AND BLOCKING CONDITIONS Th ebuffer is Tris and glycine with 20% Ethanol. This is tarnsferred at 75volts for 75minutes. The blocking agent is 5%Non-fat dried milk in trsi buffer saline -tween(0.1%)(TBS-T)

SECONDARY ANTIBODY The secondary antibody is always rabbit (HRP) from DAKO and used at 1:1000, in blocking buffer

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

WHAT STEPS HAVE YOU ALTERED? altered ocncetrtaion of primary and secondary

ANSWER:

Thank you for providing these details and I'm sorry to hear that you are experiencing difficulty with your most recent vial of ab23707. Both lots 153790 and 143130 originated from the same main batch and there should be no difference between them. It seem as if the latest vial that you received was somehow damaged and went off during the shipping process.

I would be happy to send you a free of charge replacement vial of ab23707. However, there is currently no stock left of this antibody. A new batch is in production with the release date to be confirmed.

I can set up a free of charge replacement order for you such that a vial from the new batch will be sent when it arrives into stock. Alternatively, I can provide you with a credit note or refund.

Please let me know how you would like to proceed and I look forward to hearing from you.

Our laboratory performed Western blotting on neuronal cell line culture samples extracted using RIPA, Trizol and 5X SDS. Unfortunately, we are still unable to detect the 66 kDa PINK1 protein. Only the truncated 33 kDa was detected.

ANSWER:

Thank you for your enquiry.

Charlotte is absent for the festive break. I am handling her enquiries in her absence.

I have read through the correspondence that you have been having with her. I am sorry to hear that you have been having difficulties detecting the 66KDa PINK1 band. I can only suggest that the cleavage product is more prominent than the 66KDa product.

I ask, could it be that your cultured cells do not have the 66KDa product and only have the 33KDa cleavage product? It may be worth trying a positive control tissue lysate to determine this possibility.

I would like to offer you a credit note given the optimisation that you have performed. This is provided that the antibody was purchased within the past 90 days. For me to do this please can you e-mail me details of the date of purchase and purchase order number.

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

Suzanne would like to know details of the postive control of ab23707. How was the liver lysate prepared? Is it a particulate or membrane preparation?

ANSWER:

Thank you for your enquiry.

I am sorry that my response has taken so long. I have found it difficult to obtain a response from the source of this antiserum.

The following protocol is what was used to homogenize the murine liver lysate which is used as a positive control for PINK1 Polyclonal Antibody. The tissue was prepared as a membrane preparation.

General Tissue Preparation for Western Blot:

1. Place tissue in lysis buffer (recipe below*) 2. Homogenize ~ 2 mins. 3. Centrifuge ~ 1300 x g for 20 mins. 4. Decant and reserve supernatant for later testing. 5. Resuspend cell pellet in desired buffer for use in SDS-PAGE. 6. Bradford to determine protein concentration 7. Dilute sample 1:1 with 2X SDS PAGE Buffer 8. Boil 2 mins. 9. Chill, then ready for Western Blot.

*Lysis Buffer: 10 mM TRIS 5 mM EDTA 0.5 mM PMSF with protease inhibitors (leupeptine, chymostatin, pepstatin A, and antipain)

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

Our laboratory recently purchased the PINK1 antibody (ab23707) from your company. From the datasheet, we understand that this PINK1 antibody can react with human, mouse and rat samples. At the same time, it will detect a 66 kDa and a 33 kDa bands. According to the datasheet, we performed Western blotting using this antibody at a concentration of 4 mg/ml as recommended on mouse tissue samples and human cell lines. The results are attached. We will like to make a few enquires:

* Why is the 66 kDa protein band unable to be seen in the control samples but seen in the apoptotic samples? * How does the 33 kDa cleavage fragment comes about? * Why is there an additional band below the 33 kDa protein band?

ANSWER:

Thank you for your enquiry. I am sorry to hear that you are having problems with this antibody.

In response to your questions: I have to ask, please can you confirm that the molecular weight indicators (66KDa and 33Kda) are in the correct position. Are you running a low molecular weight ladder (<100KDa). Upon seeing your blot for the first time it appeared that your two bands that are common to all of your samples may indeed be the 66kda and 33kda cleavage product. It may be that your apoptotic cells are producing an additional higher molecular weight cross reacting band.

Unfortunately I do not have details of how the 33KDa cleavage product comes about. However, the truncated form of the protein is referred to in: Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16899-903 (PubMed reference 12477932).

Should my comments fail to resolve your enquiries and you feel you require technical assistance, please do not hesitate to contact our technical team. It would help us, should you deem necessary if you could fill in the on line technical questionnaire to give us the best possible chance of resolving your problem.

http://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=23707&mode=questionaire