Anti-Alcohol Dehydrogenase antibody (ab24434)

Since the results were so uncertain, I tried only once again with a new cell line that I am sure to have both the enzymes (ADH and CYP2E1) and actually I obtained a strong band. Unfortunately not only one but many and with similar MW.

Probably I have to go further down with the concentration of the primary antibody but at the moment I have to do other experiments, so I will do it in the future months.

Moreover, I don't have fresh tissue avaiable and I used it only as a control so now that I found an alternative positive control (the cells aforementioned) I can't spend other time on the tissue.I would like anyway to thank you both for your help.

Moreover, I am beginning with immunofluorescence and I have seen that you have a new book about IHC/IF protocols for your customers. In the last 6 months we have bought at least 4 antibodies and I will need more in the future, but not before October, so I would like to ask you if it is possible to have the book anyway...

Thank you very much again.

Best regards

ANSWER:

Thank you for your email.

I am very sorry for your disappointment. I do appreciate your cooperation in this whole case which actually happened to be a strange as well as interesting. I am presenting this case to my whole department next week to know what other scientists are thinking about the problem. I will forward you the summary of the discussion. In future if you need any assistance from us we will be very happy to help.

Regarding the IHC book I am sorry only the new secondary antibodies orders are eligible for this book. This is a good book so I would suggest buying the antibody now and using it within 6 month time period which is the Abcam product guarantee period. In case antibody fails you can always call us for free of charge replacement.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

LOT NUMBER GR-42219 ORDER NUMBER 911977

DESCRIPTION OF THE PROBLEM No signal only in the mouse sample, good signal in human samples

SAMPLE mouse liver homogenate and cell extracts from hepatoma cell lines

PRIMARY ANTIBODY ab24434, dilution 1:1000 in milk 5% in TBS+Tween 0.1%, overnight (15 hours) at 4C, 3 washes 10 min each

DETECTION METHOD Immobilon Western Chemiluminescent HRP Substrate (Millipore cat no. WBKLS0100)

POSITIVE AND NEGATIVE CONTROLS USED positive control: mouse liver homogenate, negative control Huh-7 cell line

ANTIBODY STORAGE CONDITIONS aliquoted and stored -20C

SAMPLE PREPARATION Lysis buffer (liver): NaCl 150mM, TRIS 10 mM, EDTA 1 mM, EGTA 1 mM, Tryton X100 2% (pH 7.4)+ Protease inhibitor cocktail (sigma P8340)

Lysis buffer (cells): NaCl 150mM, TRIS 50 mM, sodium deoxycholate 0.1%, SDS 0.5%, NP-40 1% (pH 7.5)+ Protease inhibitor cocktail (sigma P8340)

AMOUNT OF PROTEIN LOADED 30 microg

ELECTROPHORESIS/GEL CONDITIONS SDS PAGE Tris-Glycine, 12% Acrylamide

TRANSFER AND BLOCKING CONDITIONS transfer Tris-glycine-20% methanol for 90 minutes at 0.3 A Blocking Milk 10% in TBS+Tween 0.1% for 60 minutes at RT

SECONDARY ANTIBODY anti rabbit IgG-HRP (R&D cat no. HAF008), dilution 1:5000 in 5% milk in TBS+Tween 0.1%, 90 minutes at RT, 3 washes 10 min each

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

ANSWER:

Thank you for your enquiry regarding ab24434 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

The antibody is working well with HuH7 cell line, which means there is no problem with the antibody itself. I presume there might be a problem with the preparation of tissue lysates. It would help me in understanding the fault if you could send the protocol used for preparation of tissue lysates and WB image?

Looking forward to hearing from you soon.

We have a question about ab24434 (Alcohol Dehydrogenase antibody): With which type of ADH does this antibody react (I, II or III) ? Do you have special literature (references) for this antibody, maybe with an example of a WB figure?

ANSWER:

Thank you for your enquiry.

The immunogen was used is ADH type I. I have updated the datasheet with this information. We do not know the cross-reactivity to type II and type III. The originator is looking into obtaining a Western blot image. If I receive this, I will forward it along to you.

I hope this information helps. Please do not hesitate to contact us if you need anything further.

ANSWER:

Thank you for your enquiry.

I have been in touch with the source of this antibody and I can tell you that the immunogen used to raise this antibody was purified yeast ADH.

I am sorry that I cannot be more specific with the specificity of this antibody this is unknown by the originator of this antibody.