Anti-HA tag antibody [4C12] - ChIP Grade (ab1424)

Our lab is trying to use these antibodies (ab9110 and ab1424) to do immunohistochemistry on sections from various mouse organs. We were wondering if these antibodies are tested and approved for use on paraffin sections only, cryopreserved sections only, or both?

ANSWER:

Thank you for contacting us.

Both antibodies have not yet been tested in any IHC application (neither IHC-P or IHC-Fr).

We do have a testing discount program that you can use in that case. Here is some information about how the testing discount program works:

The testing discount program is for customers who like to use an antibody/protein on an untested species/application. You would purchase the antibody at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody at the full price (100%) of the antibody you have tested. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

I'd be happy to issue you a discount code for one or both antibodies. Please let me know.

Also, please do not hesitate to contact us if you need any more advice or information.

What is the immunogen?

ANSWER:

Thank you for your enquiry.

The immunogen was YPYDVPDYAC.

Please let me know if you have any more questions.

I attached the protocol that I used. Actually, My VSVG antibody (catalog number:ab18612-200) that we ordered together with those two antibodiies worked when I used this protocol.

Thank you so much.

ANSWER:

Thank you for your enquiry. Jen is out of the office, so I am answering her correspondence.

I understand that you used a third antibody successfully in this protocol, but each antibody is different and sometimes must be optimized to work well.

What is the specific problem you are getting with each antibody? Are you seeing no signal whatsoever, high background, etc?

What blocking solution did you use? 5% milk? BSA?

How long and at what temperature did you incubate with the primary antibody? The protocol you included said 2-3 hours at room temperature or overnight at 4 degrees.

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

Customer called about problems with this antibdy in ICC and WB - no signal. Customer has used this antibody many times in the past under the same conditions with good result. This he also used a different company's HA tag ab for comparison and that worked.

Order# 86044.

ANSWER:

Thank you for your phone call and I'm sorry to hear that you are experiencing difficulty with this antibody. We have not received any other complaints regarding this batch and so it's a possibility that this latest vial which you received went off at some point during the shipping process. I have arranged for a replacement vial to be sent to you free of charge. This is on order# 87816 and you should receive this Wednesday. Please let me know how the replacement works out for you and if you have any additional questions.

Here are some answers to your questions:

1. Please describe the problem (high background, wrong band size, more bands, no band etc).---Non specific bands all over the blot 2. On what material are you testing the antibody in WB? Species? Cell extract/ Nuclear extract?--- cell lysates from BHK cells Purified protein? Recombinant protein?

3. How much protein did you load?--Loaded around 10ul of lysate, did not quantify. How did you prepare the lysate for the analysis (protease inhibitors etc)?---Lysates were prepared using 1%DM (dodecy maltoside) with protease inhibitor cocktail. Did you heat the samples?--No, its a membrane protein, and heating would disrupt it

4. Primary Antibody Specification (in which species was it raised against)?--Mouse HA tag (Abcam) At what dilution(s) have you tested this antibody?--1:1000, 1:10,000 Incubation time, wash step?:1hr followed by 3 washes for 5min each 5. Secondary Antibody Specification (in which species was it raised against)?--goat anti mouse IR labbled At what dilution(s) have you tested this antibody?: 1:5000 Incubation time, wash step?--45min followed by 3 washes for 5min each Do you know whether the problems you are experiencing come from the secondary?---its not a problem of secondary, all my blots are fine under similar conditions with same secondary 6. What detection method are you using?--Odyssey scanner bwith detects IR labelling

7. 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? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4?C with agitation)---the odyssey blocking buffer is recommended for 1hr at 37degC

Are your washing steps sufficiently stringent? ---we use 3 five minute washes after primary and secondary

At what size are the bands migrating? Could they be degradation products of your target? --they all are non-specific

Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient)

8. Optimization attempts How many times have you tried the Western?--4X Do you obtain the same results every time e.g. are background bands always in the same place? yes What steps have you altered?--different antibody dilutions 9. Did you apply positive and negative controls along with the samples? Please specify.--no

ANSWER:

Thank you for providing me with details regarding your protocol and also for the image you submitted. We have had some good feedback regarding this antibody in Western blotting, and customers have reported seeing a clean signal using ab1424 at a dilution of 1:1000. Since the extra bands that you are seeing are not due to your secondary, and I don't think diluting the primary any further is going to help, I can offer you a replacement vial of this antibody to try.

What was the lot number that you received (it is located on the vial)? Also, what was the Abcam order number or purchase order number that was used for your order for ab1424?

Thanks again, and I look forward to hearing from you.