Anti-MiTF antibody [21D1418] (ab13703)

Hello, what is going on? First you told me that the delivery of the C5 ab is delayed until September, now I had to realize that the ab I ordered instead, ab13703 Mouse monoclonal [21D1418] to MiTF does not work at all!!! I tried this ab on WB of lysates from 501 mel cells and 293 cells transfected with an Mitf construct in dilutions from 1:1000 to 1:250 over night and it didn't show any bands at all!!! Controls showed that conditions and secondary abs worked fine. This is really very disappointing and I hope I get an refund.

ANSWER:

I'm very sorry to hear you are experiencing problems with ab13703 and for the unexpected delay in the production of the C5 clone.

I understand you are disappointed, you mention controls, are these nuclear lysates of A375 or Raw cells? These are our positive controls shown to contain detectable levels of MiTF. If you have tested those and you did not detect any signal please let me know your order details and I will immediately arrange for a refund with our accounts department.

If you have not run these positive controls, the reason for the lack of signal in your cells could be that the levels of MiTF are below detection level. I would therefore suggest to run the positive controls and if you have not done a nuclear extraction I would also suggest trying this, as it will concentrate the protein in your samples.

I hope these suggestions will help, please be assured that if the antibody does not work in the positive controls I will arrange for a refund,

1. Order details: • Batch number: 90067 • Abcam order or Purchase order number: • Antibody storage conditions (temperature/reconstitution etc) 4oC

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

3. On what material are you testing the antibody in WB? • Species: Human • Cell extract or Nuclear extract: A375 and malmie cell extract • Purified protein or Recombinant protein:

3. The lysate • How much protein was loaded: 30µg • What lysis buffer was used: Hepes NCL with phosphatase inhibitors, Protease inhibitor cocktail and DTT • What protease inhibitors were used: Sigma human cocktail • What loading buffer was used: Nupage 4X LDS • Did you heat the samples: temperature and time: 100oC 5min

4. Electrophoresis/Gel conditions/ Transfer conditions • Reducing or non reducing gel: non reducing • Gel percentage : 4-12% • Transfer conditions: Nupage transferr buffer 20% methanol, 2 hours 30V 5. Blocking conditions • Buffer: 1X PBST • Blocking agent: milk, BSA, serum, what percentage: 5% milk • Incubation time: overnight • Incubation temperature: 4oC

6. Primary Antibody • Specification (in which species was it raised against): Mouse • At what dilution(s) have you tested this antibody: 1:250 • What dilution buffer was used: 5x marval PBST • Incubation time: 1 hour • Incubation temperature: Room temp • What washing steps were done: 2 X 5 min 2X 15 min

7. Secondary Antibody • Specification (in which species was it raised against)? Rabbit • At what dilution(s) have you tested this antibody: 1:5000 • Incubation time 1 hour room temp • Wash steps: 2 X 5 min 2X 15 min • Do you know whether the problems you are experiencing come from the secondary? they do not

8. Detection method ECl, ECl+, other detection method: ECL and ECL PLUS

9. Background bands • Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): no bands at all • 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? • Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

10. Did you apply positive and negative controls along with the samples? Please specify. used A375 lysate the same as in your literature 11. Optimization attempts • How many times have you tried the Western? once • Do you obtain the same results every time e.g. are background bands always in the same place? • What steps have you altered?

ANSWER:

I'm very sorry to hear you are having problems with ab13703, thank you for providing your protocol details, these are very useful.

I would like to recommend the following modifications to your protocol: -I would not recommend using DTT in the lysis buffer. Please consider using an NP-40 buffer (RIPA buffer) and make sure the cocktail of protease inhibitors is fresh.

-please make sure the loading buffer you purchase is reducing (DTT, SDS, beta-mercaptoethanol should be present)

-separate the protein in reducing conditions

-block the membrane in 5% milk for 1hr at RT (in TBST)

-incubate the antibody overnight in TBST (no milk)

-you may need to consider nuclear extraction if the levels of MiTF are very low in your samples

I hope these suggestions will help, please do not hesitate to contact us again if you need further advice,

In answer to your questions:

1) I do not know the batch number as I inadvertently discarded the original tube following immediate sub aliquoting for storage at -20oC (ooops!).

2) The secondary antibody for the MITF was used successfully on the same blots for other proteins. In addition the secondary antibodies are shared across the lab and any problems would therefore be readily apparent.

As regards replacements we would like to try the C5 mouse monoclonal to MiTF (ab 12039) which has been used successfully by another lab and reviewed on your website.

I will let you know how we get on with this and "advertise" any success we have on your website

ANSWER:

Thank you for your e-mail.

I have placed a new order for you one vial of ab12039 - free of charge. Please do let us know how you are getting on with this alternative antibody.

Good luck with your research!

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 36012064

DESCRIPTION OF THE PROBLEM No signal or weak signal

SAMPLE Whole cell lysates from the following human cell lines: A357 (positive control) SKmel 28 (Melanoma) WM266.4 Melanoma HT29 (colon Ca) HCT116 (colon Ca)

Expression is based on Cancer Cell Dec 2004 vol 6: 565-576.

PRIMARY ANTIBODY 1o Ab 13703 used at 1:200 dilution in 3% Marvel TBST. Incubated on rollers with signal surface in contact with Ab solution, at 4oC o/n (16h). Washed twice in 10-20ml TBST x 10 min.

SECONDARY ANTIBODY Incubated in 2o Ab goat anti-mouse (Bio-Rad) at 1:5,000 in 3% milk TBST at RT x 1h.

Washed four times in 10-20ml TBST x 10 min.

DETECTION METHOD ECL detection Pierce Supersignal west pico as per manufacturers instructions.

POSITIVE AND NEGATIVE CONTROLS USED A375 (positive in your hands negative in CC Dec 2004!!!!! qv) SKmel 28 (Positive in CC Dec 2004) HT29 Colon cell line expected to have low expression qv CC Dec 2004 HCT116 Colon cell line expected to have low expression qv CC Dec 2004

ANTIBODY STORAGE CONDITIONS Antibody Stored in 20ul aliquots at -20oC

SAMPLE PREPARATION Lysed in NaCl-HEPES buffer PH 7.5, containing 0.3%NP40, one Complete (Roche) inhibtor tablet per 10ml, NaVO4, NaF, DDT. All procudures carried out at 4oC (on ice. Samples lysed on ice x 15 min, spun at 4oC and supt recovered. BCA protein determination carried out on all samples.

AMOUNT OF PROTEIN LOADED 50ug total lysate loaded per lane.

ELECTROPHORESIS/GEL CONDITIONS 10% Tri-Gly 15 well precaste NOVEX gels, run under standard conditions.

TRANSFER AND BLOCKING CONDITIONS Transfered at 100V x 90min to PVDF membrane. Transfer confirmed with R250 staining. Membranes blocked at room temp in 3% Marvel in Tris buffered saline containing 0.1% Tween 20 (TBST) x 1h.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? We are attempting to buy another antibody from a referenced source (C5)

ADDITIONAL NOTES We have had two people independently repeat this experiment with a variety of melanoma cell lines with negative results. In addition a number of other proteins have been successfully probed on the same blots (blot cut into sections and MITF always probed using virgin blots).

If upon examining these data you deem Ab 13703 to be causally related to these observations then we would consider evaluating ab2384 (a mixture of C5 and D5 preps) as a replacement.

ANSWER:

Thank you for your e-mail and for taking your time to fill in the on-line Questionnaire. I am very sorry to hear that you are having problem with this antibody.

I would be grateful if you could give me the batch number you have tested.

1. Have you tried to prepare nuclear extract? As you probably know MiTF is a nuclear protein.

2. I understand that you have managed to detect other proteins on the same blots. Have you actually used and tested the same secondary antibody? Does the detection system work properly?

I could offer you either a new vial as a free of charge replacement or a reimbursement. Please do let me know how you would prefer to proceed.

I am looking forward to hearing from you soon.

Unfortunately, I reapeted the western blot twice with the new Ab and still do not get any binding of the primary. I used A375 cytosolic and nuclear extracts as control as well as another melanoma cell line believed to produce MITF. For the first western blot, I used three different primary Ab dilutions: 1:1000, 1:500 and a no-primary Ab control. I also used three different sample dilutions: 40, 20 and 10 ug of proteins. For both melanoma samples, I detected a faint band between 50 and 60 KDa in the nuclear extract only after a very long exposure using the ECL system (35-40 minutes). Unfortunately, the same band is detected in the control (no-primaqry Ab). I repeated the western blot a second time using a 1:250 primary Ab dilution and got the same result. The band detected around 55 KDa could partially be MITF but since is is detected in the no-primary control, it is hard to distinguish from background. I am using ab6728, a rabbit polyclonal to mouse IgG, which works fine with other primary Abs. I could try to use a different secondary Ab to see if the ~55KDa band is still detected. Would you have any other suggestions?

Thank you for your help.

ANSWER:

I have received the following information from the source of ab13703:

"We reviewed the QC data for this antibody and it is very clean in our hands. QC data for MCF7, T98G, Daudi, A375, and Raw all reveal 1 clean single band at the correct molecular weight (around the 55 kDa marker). The bands are strong and robust. In looking through the customers note, we noticed that the customer mentions that both cytosolic and nuclear fractions were used. The lysate preparation may have affected the results. We have not tested the antibody in fractionated lysate. We use whole cell lysate. I would recommend to the customer to use whole cell lysate."

I hope this information will help, please let me know if I can be of further assistance,