Anti-TIMP2 antibody [3A4] (ab1828)

Hi, I’m trying to find antibodies to detect canine TIMP-2 and MMP-14 and was wondering whether the products I list below are predicted to cross-react with the canine protein. I’ve attached alignments which I hope will be of some help to determine cross-reactivity. Anti-TIMP2 antibody [3A4] (ab1828) Anti-MMP14 antibody [113-5B7] (ab58815) Anti-MMP14 antibody - Hinge region (ab38971) Kind regards /

ANSWER:

Thank you for contacting us. According to the alignment information that you have sent to me it looks like you should have success with using these antibodies with canine protein. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.  

A replacement would be great and then I will likely order a large amount in the future.  Do I need to place an order or will you place the replacement order? Thanks so much for your help!  

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

I tried both TIMP antibodies at 1:1000, 1:500, and 1:100. I only got them to work at 1:100 with an overnight exposure. The control antibody I used that detects viral protein was easily detectable. I didn't quantify the amount of protein that I loaded, but I loaded the maximum 15ul volume. I included other samples on the same gel prepared the same way and they were easily detectable via WB using other antibodies.

Block: 5% milk, .1% Tween-20 in PBS Wash: 1% milk, .1% Tween-20 in PBS Blocking and AB exposure

1. Trim and mark ladder 2. Wash the membrane in PBS 3. Block 2x 40' RT 4. Wash 2x 5 min in wash 5. Incubate in primary AB diluted in wash O/N 4 degrees 6. Wash 4x 10min in wash 7. Incubate in secondary AB diluted in wash 1 hr RT 8. Wash 4x 10 min in wash 9. Place in PBS 10. Develop

I also tried ICC on transfected cells where both antibodies only worked at a 1:10 dilution with the following protocol: 1. Wash in PBS 2. Fix in 4% PFA in PBS 15' RT 3. Wash 3 times in PBS 4. Incubate in blocking solution for 15 min at RT 5. Wash with perm sol. 6. Incubate in perm sol. 15 min at RT 7. Incubate in primary antibody (dilute in perm sol) for 1.5hrs at RT 8. 3x 5 min washes in perm sol. 9. Incubate in secondary antibody for 1.5hrs at RT (dilute in perm. sol.). Cover with foil to protect from light from this point on. 10. 3x 5 min washes in perm sol. 1 1. Incubate in DAPI 1:10,000 in PBS for 10 min at RT 12. Wash with PBS 13. Add vectashield and mount Blocking solution PBS with 3% BSA and .01% sodium azide Permeablization solution PBS with 1% BSA, .1% TritonX-100, .01% sodium azide

ANSWER:

Thank you for taking the time to send me your protocols relating to this issue. I have been able to review them and agree that the problem here is the product.

I would like to offer you free of charge replacements for ab1828 and ab61224. I may also offer you a credit note/refund that may be used against this or any outstanding invoices.

As you have mentioned before that you will be using alot of this antibody for future experiments, I thought I might inform you that Abcam will discount orders above 5 units.

Please let me know how you would like to procceed and do not hesitate to contact us with further questions or if there are other ways in which we might help you reach your research goals.  

I purchased a TMIP-2 antibody (ab1828) where the recommended WB

concentration was 1:1000. I was only able to detect TIMP-2

overexpressed in cell lysates using the antibody at 1:100

concentration.

I also purchased a TIMP-1 antibody (ab61224) where the recommended WB

concentration was 1:500-1:1000. I was only able to detect TIMP-1

overexpressed in cell lysates using the antibody at 1:100

concentration and the antibody did not detect endogenous TIMP-1 in 293

cells (the suggested positive control).

Since the antibodies did not work as well as suggested, would it be

possible for me to order them again at a discounted price since I have

to use so much antibody for it to

ANSWER:

Thank you for contacting Abcam in regards to ab1828 and ab61224. I am sorry that you found that these products do not perform as well as expected in your applications. 

I would appreciate it if you could provide me with protocol details of the approach that you are using so that I can better determine potential reasons for the reduced performance of these antibodies. I am also very interested in the method of sample preparation that you have used. I would like to offer you assistance if possible for resolving these issues in your experiments. Could you please send me the protocols that you have used for each?  

Could you also send the lot numbers for the antibodies and a order number or PO ?

Could you also confirm the species of the tissue tested? If we cannot resolve these issues with you I would be happy to offer you a replacement or credit.  

The gels that we have been using are non-reducing, non-denaturing.

The amount of protein loaded vary according to our daily plans.... but should be more than sufficient in order to detect bands. We haven't used loading controls at this point... we wanted to see something first.

We used the NP-40 buffer documented in your western blot beginners pdf file.

In the case of all the antibodies ordered, they have been designated by Abcam as tested for Western blots. Do you have any images on files that I can take a look at? What type of tissue did you use for the testing? How different was your protocol from the one established in our lab? Sorry for all the questions but maybe I can use this info for some of the trouble shooting on our end.

ANSWER:

Thank you for your enquiry.

I appreciate your continued patience in this matter. I have received some feedback from some of the sources of the antibodies that you are enquiring about;

ab7033 - To follow on with the comments that I have made the source of mouse monoclonal [MMP2/2C1] to MMP2 (ab7033) was also concerned with regards the approach that you have used for your sample preparation. I appreciate that you have been using an NP40 buffer extraction. However, this is an approach designed for a cytoplasmic preparation of cell culture cells. I would like to follow up my previous email by recommending that you perform a loading control experiment using an antibody that targets a "housekeeping protein" for example GAPDH or beta actin. This can be performed under the conditions best recognized by the antibody you are using; most likely denaturing, reduced. This will enable you to fully determine the integrity of your protein with respect to its protein composition. My biggest concern with the blot images that you have provided me with are the band doublet that you have been detecting either side of the 37KDa marker as this is present in the majority of your blot images.

The source of ab6586 - Collagen IV antibody (ab6586) makes a similar suggestion although Collagens should in fact be electrophoresed as you have been using non-denaturing, non-reduced conditions. However, it is important that the integrity of your samples are confirmed.

I am still awaiting further information and am in the process of requesting blot images for the antibodies that you have enquired about. I appreciate your continued patience.

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