Anti-Exonuclease 1 antibody [266] (ab3307)

Some troubles with ab3307.

Could we get a detailed protocol from you, please. We have some general questions, the answers to which will enable us to investigate this matter as quickly as possible.

a.. Exonuclease 1 antibody [266] (ab3307) b.. Antibody store at 4 ?

1. Order details: a.. Batch number:98442 b.. Abcam order or Purchase order number: 73965 c.. Antibody storage conditions (temperature/reconstitution etc) 4?

2. Please describe the problem (high background, wrong band size, more bands, no band etc). No expected band

3. On what material are you testing the antibody in WB? · Species: Human GBM cell line

· Cell extract or Nuclear extract: nuclear extraction buffer.

· Purified protein or Recombinant protein: recombinant protein

3. The lysate a.. How much protein was loaded: 50mg What lysis buffer was used: 1x Pharmigen-Protease - CocktailNuclear extraction buffer (NEB): 25mM Hepes pH 7.5, 500mM NaCl, 1mM DTT, 10mM NaF, 10% Glycerol, 0.2% NP40, 5mM MgCl22ml

a.. What protease inhibitors were used: Roche protease cocktail

a.. What loading buffer was used: SDS loading buffer

a.. Did you heat the samples: temperature and time: (only sample buffer) 95?/ 5min

4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: b.. Gel percentage : 9% c.. Transfer conditions: 1.2mA/cm2

5. Blocking conditions a.. Buffer: TBST b.. Blocking agent: milk, BSA, serum, what percentage: 5% BSA c.. Incubation time:1 hour d.. Incubation temperature: room temperature

6. Primary Antibody a.. Specification (in which species was it raised against): mouse · At what dilution(s) have you tested this antibody: 1:100

· What dilution buffer was used: blocking buffer 5% BSA TTBS

· Incubation time: overnight

· Incubation temperature: 4?

· What washing steps were done: TBST 5 min/time * 3 times

7. Secondary Antibody a.. Specification (in which species was it raised against)? mouse b.. At what dilution(s) have you tested this antibody: 1:2000 c.. Incubation time: 1 hour d.. Wash steps: TBST 5 min/time * 3 times e.. Do you know whether the problems you are experiencing come from the secondary? Other monoclonal antibody is OK.

8. Detection method ECl, ECl+, other detection method: ECl

9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): yes

· Is the blocking step sufficient? yes

· Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes

· At what size are the bands migrating? Could they be degradation products of your target? There is no band

· Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) there is no band.

11. Did you apply positive and negative controls along with the samples? Please specify. I have confirmed its mRNA expression by microarray and real-time PCR. 10. Optimization attempts · How many times have you tried the Western? 2 times

· Do you obtain the same results every time e.g. are background bands always in the same place? yes

· What steps have you altered? No

ANSWER:

Thank you for your enquiry. We are very sorry to hear that your are having difficulty with this antibody.

Here I am sending you our protocol for WB. A. 1. Lyse the PBS-washed cell with 5% SDS, containing 5 mM iodoacetate,1 mM PMSF. 2. Sonicate sample briefly or pass it several times through a 21-gauge needle. 3. Centrifuge sample at 4oC for 10 minutes to pellet insoluble material. 4. Determine protein concentration and dilute protein sample in 1 x SDS sample buffer (125 mM Tris pH 6.8, 2% SDS, 10% glycerol, 0.0005% bromophenol blue, 10 mM DTT). 5. Denature proteins by boiling sample for 3 minutes.

B. ELECTROPHORESE ON SDS-PAGE GEL 1. Follow standard protocol. 2. Load 50 ug/lane of protein.

C. TRANSFERRING PROTEINS FROM SDS-PAGE GEL TO BLOT 1. Make a sandwich composed of filter paper-gel-blot (PVDF or nitrocellulose)-filter paper, and place in electric field where negatively charged proteins migrate out of gel onto blot. Apply current (60-70V) according to recommended time.

D. BLOCKING THE BLOT 1. Remove blot from transfer apparatus and immediately place into blocking solution (5% non-fat dry milk with PBS + 0.1% Tween 20) and block according to recommended time and temperature (20-30 minutes at room temperature with agitation).

E. BINDING OF PRIMARY ANTIBODY TO TARGET PROTEIN 1. Dilute antibody in 5% non-fat dry milk with PBS + 0.1% Tween 20 according to suggested dilution on data sheet (1:50 of 0.2 mg/ml). For purified antibodies, final concentration is generally 1-3 ug/mL. 2. Decant blocking solution and add antibody solution. 3. Incubate with agitation at room temperature for 2 hours at room temperature (or overnight at 4oC). 4. Wash blot with wash solution (PBS + 0.1% Tween 20) for 30 minutes with agitation, changing wash buffer every 10 minutes.

F. APPLYING SECONDARY ANTIBODY TO BLOT (LINKING) 1. Incubate blot with diluted (1:2500 in 5% non-fat dry milk with PBS + 0.1% Tween 20) and labelled secondary antibody at room temperature for 1 hour with agitation. 2. Decant secondary antibody solution, add wash solution (PBS + 0.1% Tween 20), and wash with agitation for 30 minutes, changing wash solution every 10 minutes.

G. DEVELOPING BLOT (LABELING) 1. For colorimetric detection, add enzyme substrate to blot. 2. Incubate at room temperature until satisfactory color development is obtained. Stop reaction by washing blot in water. Dry at room temperature. 3. Chemiluminescent detection, follow the instructions provided by manufacturer

We hope that this information will be useful!

Thanks for your help. I would like the MCF-7 positive control sample to try please. I have sent the full details of my western blot procedures before. Thanks again

ANSWER:

Thank you for your reply.

This is to let you know that I have just placed a new order for you one vial of ab14860 (MCF-7 nuclear lysate) - free of charge.

Please do let me know how you are getting on with the ab3307.

Good luck and have a nice weekend!

I am writing regarding the exonuclease I antibody (ab3307). I am not happy with this antibody at all. I am currently using the 2nd replacement (Lot 94880) you have sent me, as i got no signal with either of the first two batches. I ran some samples on a gel and i include the western as an attachment. I did manage to get a weak signal which looks a bit larger than the predicted molecular mass, with commercial HNE, there was 10ug of protein in this lane. I left the film exposing for 40 minutes to get this signal. My samples, which included Hela whole cell extracts, approx 2x10^4 cells per well, and extracts which i have hopefully depleted ExoI by RNAi, are also on the gel. I got no signal from these at all. I used the primary antibody at 1/100 overnight. I know the transfer and secondary procedures are working as I regularly carry out western blots. This antibody really doesn't work well enough for me (or I imagine) anyone to use. I first ordered this antibody months ago now and have spent a lot of time trying different blots and antibody batches. I really need a working antibody to know if the RNAi is working. Please could you get back to me soon so we can get this sorted asap.

ANSWER:

Thank you for your e-mail and your phone call. I am very sorry to hear that even the second vial which we have sent to you as a free of charge replacement did not work.

I have looked trough your previous messages which are stored in our computer system and see that one of my colleagues Miss Jennifer Priester has tried to help you in the past.

I have been trying to find some details of your Western blot assay but unfortunately can't see if we have ever received the completed Western blot Questionnaire from you. So I am trying to gather the information from your previous letters.

This target is a nuclear protein so it is extremely important to prepare nuclear extract and use it rather than whole cell lysate. Unless, the whole cell lysates are prepared from relatively high cell number. Unfortunately, we do not know how Exo1 is expressed in HeLa cells. Is it expressed at high or low levels? All we know is that this product has been tested and characterized for Western blot using MCF-7 and MAD109.

As I understand from you last e-mail, you used 10 ug of HNE. As the product datasheet states, mouse recombinant full length protein was used to raise this antibody. Although, ab3307 does recognize human Exo 1 (as well as mouse), it is very likely that the human protein is processed differently from the mouse therefore the band size may be different. It is also important to emphasize that recombinant and not native endogenous protein was as immunogen. Therefore, it would be perhaps a better idea to run recombinant mouse Exonuclease as positive control along with the samples.

I have no information if you have actually used RIPA buffer for lysate preparation. We would normally advise our customers to use this buffer in order to prevent any degradation of the target protein.

RIPA Base Ingredients Tris-HCl: 50 mM, pH 7.4 NP-40: 1% Na-deoxycholate: 0.25% NaCl: 150 mM EDTA: 1 mM PMSF: 1 mM Aprotinin, leupeptin, pepstatin: 1 microgram/ml each

RIPA Protease Inhibitors Phenylmethylsulfonyl fluoride (PMSF) (200 mM stock solution in isopropanol; store at room temperature) EDTA (calcium chelator; 100 mM stock solution in H2O, pH 7.4) Leupeptin (store frozen in aliquots, 1 mg/ml in H2O) Aprotinin (store frozen in aliquots, 1 mg/ml in H2O) Pepstatin (store frozen in aliquots, 1 mg/ml in methanol)

You used HeLa whole cell extracts, approx 2x10^4 cells per well. Have you actually determined the protein concentration of these samples. We would recommend loading 25-30 ug protein per lane. It may well be that you would need to increase the cell numbers to get a proper signal.

I am very sorry to hear that you have been struggling with this antibody and find it very difficult to get good results with it. I have looked at our catalogue and actually we have MCF-7 nuclear extract code number: ab14860, so I could offer one vial free of charge for you to test. Please take a look at the product datasheet:

http://www.abcam.com/index.html?datasheet=14860

I would like to emphasize that we try to help our customers as much as possible but do not regularly offer positive control for testing an antibody. However, I do understand that it has been very frustrating for you so I really would like to help you more.

Please do let me know how you would prefer to proceed. I am looking forward to hearing from you soon.

Thanks for the replacement antibody. I have tried it and am still failing to get it to work. I ran commercial HeLa nuclear extract on a gel at different diltuions, the largest amount contained 100ug protein, and used the antibody for 2hrs at room temp at a 1/50 dilution. It does detect a weak band at about 60kDa but nothing around 90kDa. As far as i can see there are only two possibilities for why i got no signal: 1) the antibody doesn't work, but if others have used it that seems unlikely 2) the protein is not expressed or expressed at low levels in HeLa cells - do you have any paper references where this antibody has been used? Thanks for your help

ANSWER:

Thank you for your email. Part of the problem is that I think that you were sent the wrong batch of ab3307 as a replacement. So I'm sending you the newest one that we have received from the originator, it is on order# 72817.

I also checked with the originator and they are unaware of what the expression of Exonuclease 1 is in HeLa cells. As I previously mentioned, it is recommended to use MCF-7 cells and MAD109 cells as a positive control. Unfortunately, we are not aware of any publications that feature the use of this antibody.

Please let me know how this new batch works out for you and if you have any additional questions.

I know that my secondary antibody and transfer are working. You suggest MCF-7 cells and MAD109 cells as a positive control. Are there any other cell lines I can use? I will run some HNE, but I do need it to work on whole cell extracts in the end. Is it expressed at particularly low levels in HeLa cells? Can you recommend other cell lines?

BATCH NUMBER 90942 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No signal or weak signal - I do not see a band of the correct size, i see one very weak band of an incorrect size.

SAMPLE I have used HeLa WCE, and have loaded about 2x10^4 cells per well

PRIMARY ANTIBODY I have used the ab at 1/100 overnight. It is a mouse monoclonal against ExoI. I washed 3 times in TBS/T between primary and secondary and after secondary before development. I used it diluted in milk again

SECONDARY ANTIBODY Used in milk, DAKO anti nouse HRP conjugate at 1/4000 for 1hr at room temp

DETECTION METHOD ECL, have exposed the film for up to 2hrs

POSITIVE AND NEGATIVE CONTROLS USED none

ANTIBODY STORAGE CONDITIONS Kept at 4C

SAMPLE PREPARATION I just boiled the samples directly in SDS loading buffer (made with b mercaptoethanol) for 15 minutes

AMOUNT OF PROTEIN LOADED 2x10^4 cells per well

ELECTROPHORESIS/GEL CONDITIONS reducing gel, 10%

TRANSFER AND BLOCKING CONDITIONS Transfered for 2hrs at room temp with ice block, blocked for 1hr at room temp in 5% non fat 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? I have increased the primary concentration and transfer time

ANSWER:

Thank you for the details that you have provided. I have checked with the originator of this antibody, and they have had one other complaint regarding the batch that you received. If you would like, I can offer you a replacement batch free of charge. Alternatively, if you would prefer a refund, please let me know.

The only positive controls that we're aware of are MCF-7 (human breast cancer) or MAD109 (mouse lung cancer cells).