Anti-TIMP1 antibody [102D1] (ab1827)

had a problem with one antibody.
I attached the questionnaire below.
Best regards

ANSWER:

Thanks for completing and submitting the IHC questionnaire. I'm sorry to hear this vial of ab1827 is giving you trouble.

Your protocol looks good.One idea is perhaps a 48hr fixation time is too long, perhaps over fixing the samples and obsscuring the epitopes. Samples vary but ypically 24 hrs is the longest fixation period recommended. I would also suggest trying a high pH retrieval buffer, such as ph9.

I look forward to hearing if these suggestions prove useful.

What is the immunogen sequency for ab61224?

In regards to ab1827, is the whole TIMP1 human protein used as the immunogen?

ANSWER:

Thank you for contacting Abcam.


Regarding ab1827 the full length protein was used as an immunogen.


Regarding ab61224, it is a peptide from the central region of the protein. The specific sequence is proprietary.


I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

I need to be informed about the imunogen sequence used to obtain the antibody Abcam herein specified:
Anti-TIMP1 antibody (ab61224)
Concerning Anti-TIMP1 antibody [102D1] (ab1827) is it against the whole sequence of human TIMP1?
Thanks for attention.
Sincerely yours,

ANSWER:

Thank you for contacting us.

The immunogen for ab61224 correspond to thecentral region of human TIMP1. The full protein sequence can be found by clicking the link; http://www.uniprot.org/uniprot/P01033

The ab1827 is against the whole sequence of human TIMP1. The exact epitope of this antibody hasn't beendetermined yet.

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

Dear Sir:

We purchased antibody ab1827 for immunocytochemistry study to analysis protein expression in human clinical breat cancer samples. We tried many times based on the method provided by abcam. However, we can not get any obvious positive mmunorective cells in our samples. Attached describe clearly our experimental protocol and procedure. Please hlep us to figure out in which asepcts that we still need to improve in order to get positive results. We used a lot of abcam antibodies and we love abcam's products.

best wishes for your help and support!

1. Antibody Storage Condition:
Original antibody was saved in -20C freezer. Befor experiment,
appropriate titration was prepared and diluted antibodies were kept at 4C.
Antibody did not experience repeatedly freezing and thawing steps. .

2. Please describe the problem (high background, no staining etc).
We used this antibody to detected the presence and exprssion of TIMP1
proteins in paraffin-embedded tissue samples. We performed the
immunocytochemistry staining numerous times and we can not get any
observed immounreactive cells in the stained tissue sections.

3. On what material are you testing the antibody in IHC
Our materials are fresh clinical pathological samples. The tissue samples
are fixed and embedded by paraffin. The entire embedding procedure was
done based on the published protocol.

4. How did you fix the samples?
Tissue samples were obtained fresh and then dipped in 4% paraformaldehyde
solution overnight. After removing water from tissues and lipid contect from
tissue by xyline, tissue samples were immersed in paraffin solution at
differernt concentrations. Paraffin embedding was rotuinely performed in our
lab based on tradition procedure.

5. For formalin-fixed paraffin embedded tissue: did you apply antigen retrieval
step?
Microve method. But, based on our twenty years experience, we do not think
this treatment will help that much.

6. Blocking steps:
Furing experiment, the sections were undergone a serices of procedure to
remoe wax and re-hydrate. After pretreatment, tissue sections were treated
with 2-3% hydrogen peroxide to inactivate endogenous peroxidase. After
several washed in 1XPBS, tissuse sections were inculated with 1X NGS
(normal goat serum).

7. Primary antibody
- Specification (in which species was it raised against):
The primary antibody was raised in mouse and reactive well with
human samples. We have tested abot 50 cases of human breast and
gastric cancer paraffin sections.
- At what dilution(s) have you tested this antibody:
Based on the spec sheet provided by abcam, we used 1:50, 1:100 and
1:200 in our experiments. None of them worked will. Base on thepec
sheet provided buy abcam, we speculated that this antibody has a very
low titration, which makes the detection sensitivity might not be high.
- What dilution buffer was used:
1XPBS
- Incubation time: 24-48 hours
- Incubation temperature: 4 degrees
- What washing steps were done: Washed with 1XPBS three times, each
for 5-10 minutes.
8. Secondary antibody
- Specification (in which species was it raised against): Goat anti-mouse or
rabbit ,general-purpose
- At what dilution(s) have you tested this antibody: Different diluions had
been used. 1:500-1:1000 was used in our experiment.
- What dilution buffer was used:
- Incubation time: 20˜30minites
- Incubation temperature: room temperature.
- What washing steps were done: Washed with PBS three times, each time
5-10 minutes
- Do you know whether the problems you are experiencing come from the
secondary? We have used the same secondary antibody for other
experiments and this antibody worked very well.

9. What detection method are you using?
DAB chromogen.

10. Background staining
- Please provide an image of your staining
The backfround stainining is quite low. Please see below pictures, which
are both human breast cancer samples.

11. Which detection system did you use?
We used HRP based DAB staining system for color reaction.

12. Did you apply positive and negative controls along with the samples?
Please specify.
Yes, we did. We used beta-actin antibody to evaluate the prtein expression
levels in our tissuse sections. The results appeas to be very good.

13. Optimization attempts
- How many times have you tried the IHC? About 20 times
- Do you obtain the same results every time? Yes, it is.
- What steps have you altered? Immunocytochemistry staining was done
based on the tridition experiment procedure.

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will provide us with vital information for our monitoring of product quality

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.

I apologize for the inconvenience and am pleased to offer you a free of charge replacement or credit note in compensation, providing the item has been purchased within the guarantee period of 6 months.

In order to arrange this, I would appreciate if you could confirm the Abcam order referencenumber and data of purchase.

In addition, I can suggest you may like to consider the following suggestions as a check for future experiments:

1. Try adding 0.2% Tween to the antibody dilution buffer, blocking buffer and wash buffer. This can help to keep the antibody solubilised.

2. I can suggest to consider tyring different time points for antigen retrieval. This can sometimes help to optimize results.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Dear Tech support   One of our customers is interested in an anti TIMP-1 antibody for IHC-P, IHC-Fr and WB on Mouse samples. Ab1827 has only been tested for Human, would you like the customer to order it as part of the abreview promotion and submit a review on one of the above methods?   Please advise.    

ANSWER:

As shown on the attached pdf, the homology between the human and the mouse TIMP1 proteins is only 72%. Furthermore, the epitope of ab1827 has not been determined and it is a Mouse monoclonal antibody, meaning that the staining in IHC will be Mouse on Mouse. Please inform your customer that we have an antibody that is directed against the Mouse TIMP1 : ab86482, www.abcam.com/ab86482. This antibody has been tested in WB and IHC-Fr. I can send a testing discount code for your customer to test this antibody ab86482 in IHC-P. I hope this information is helpful to you. Please let me know if your customer would like to receive a discount code for ab86482 in IHC-P or for ab1827 in Mouse.