Anti-MMP7 antibody (ab4044)

Hello,
I will forward you the email with all the order information. This antibody was
purchased in December. Please send us a replacement.

ANSWER:

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 for one vial of ab4044.

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 am doing western blotting to detect this protein in embryonic mouse brain extract. I see a very intense band between 60-70 kDa but no other bands. no background at all. But I expect the detected band to be either 18 or 28 kDa. Have you seen this sort of thing before?

ANSWER:

We have not seen a band at around 60-70 kDa for MMP7.



This may be a problem with the primary antibody. If you have ordered this vial of ab4044 within the last 6 months or so I would be happy to offer a replacement, credit or refund. So I can process your request, please provide me with the PO number or Abcam order reference number.



Thanks and I apologize for any trouble this has caused.

Dear technical support team:
This customer has already purchased ab4044 (Anti-MMP7 antibody), and after he conducted wb assay with human sample, the result shows multiple bands, therefore she wants to ask for you help to offer any suggestion to her, I also attached the image in this letter, and her experiment step as follow:
1. Order details:

Batch number: gr44293-3

Po: xx

Abcam product code: ab4044

Antibody storage conditions (temperature/reconstitution etc) Store at +4°C

2. Please describe the problem (high background, wrong band size, more bands, no band etc).
High background and no bands at 28 kDa (pro form) and 18 kDa (active form) which are this antibody recognized.
3. On what material are you testing the antibody in WB?
· Species: Human
· What’s cell line or tissue: Human liver cancer cell lines (Hep-3B and Huh7)
· Cell extract or Nuclear extract: Cell extract
· Purified protein or Recombinant protein:

3. The lysate

How much protein was loaded: 20ug

What lysis buffer was used: M-PER Mammalian Protein Extraction Reagent (Thermo Scientific Prod# 78501)

What protease inhibitors were used: PMSF, NaF, Leupeptin, Aprotinin,

What loading buffer was used: 2X SDS sample buffer containing 20% glycerol, 4% SDS, 100mM Tris pH 6.8, 0.002 % Bromophenol blue and 100mM dithiothreitol.

Phosphatase inhibitors: Na3VO4

Did you heat the samples: temperature and time: 95°C, 5min

4. Electrophoresis/Gel conditions/ Transfer conditions

Reducing or non reducing gel: Reducing gel

Reducing agent: SDS

Gel percentage : 12%

Transfer conditions: (Type of membrane, Protein transfer verified): PVDF membrane, protein marker is successfully transferred.

5. Blocking conditions

Buffer: 1xTBST buffer containing 20mM Tris, 150mM NaCl, 0.1% Tween

Blocking agent: milk, BSA, serum, what percentage: 5% non-fat milk

Incubation time: one hour

Incubation temperature: Room temperature

6. Primary Antibody

Species: Rabbit

Reacts against: Human, Mouse
· At what dilution(s) have you tested this antibody: 1:500
· What dilution buffer was used: 5% BSA in 1xTBST buffer
· Incubation time: Overnight
· Incubation temperature: at 4°C
· What washing steps were done: Wash blot 3 times in 1xTBST buffer for 5 minutes

7. Secondary Antibody

Species: Goat

Reacts against: Rabbit

At what dilution(s) have you tested this antibody: 1:10000

Incubation time: one hour

Wash steps: Wash blot 3 times in 1xTBST buffer for 5 minutes

Fluorochrome or enzyme conjugate: HRP-conjugate

Do you know whether the problems you are experiencing come from the secondary? No

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

9. Did you apply positive and negative controls along with the samples? No Please specify.
10. Optimization attempts
· How many times have you tried the Western? 3 times
· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): No
· Do you obtain the same results every time e.g. are background bands always in the same place? yes
· What steps have you altered? Concentration of primary antibody

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear your customer has had difficulty obtaining satisfactory results from this MMP7 antibody.

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

I appreciate the time spent in the laboratory and understand the 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, apart from using BSA as blocking reagent which usually gives a stronger signal. I can suggest the customer has regrettably received a bad vial.

I apologise for the inconvenience and am pleased to offer a free of charge replacement or credit note in compensation.

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

Thank you John for your reply, I will try to test ab5706 on my samples and hopefully will get better results.

ANSWER:

I have processed your request to receive ab5706 as a replacement for ab4044. Your new order number is *****. If there is anything else I can help you with, please let me know.

  Dear John, I tried the samples with new beta mercapto and boiled for ten minutes but got the same result. Do you think this lot or vial is bad? I am really hoping to check the MMP7 levels in these samples. What would you suggest for me to try? the PO number is:

ANSWER:

I am sorry that the protocol tips did not prove to be successful. I can either send you a new vial of the antibody and you could continue testing with that. Or. I could send you a different antibody to MMP7, I was thinking that ab5706, would be a suitable replacement. I would recommend trying the different antibody and seeing if you have better success with that one.