Anti-MMP7 antibody (ab5706)

The blot that the customer run with your recommendation came out with no signal at all
They wish to receive
Ab4044
Instead
Please confirm

ANSWER:

Thanks for your reply.

If you can provide the PO number or Abcam order reference number associated with the purchase of ab5706 I will be happy to arrange shipment of ab4044 as a replacement.

Read customers answere


1) The positive control in the first lane, purified MMP7 is serving as our "maker". I used PAGE markers which are in the final lane of the gel only to indicate successful transfer of the blot.



2) An abcam antibody from abcam was used very successfully under the same conditions with these samples.



3) Next time, I can lower the amount of pure MMP7 loaded. I understand the gel percentage was a bit high, but that is a separate issue from the problem here with the antibody.

ANSWER:

Thanks for the additional information!

I look forward to seeing your next western blotwhere:

1. Lesspure MMP7 is loaded as control

2.Molecular weight markers are noted

I have attached the image.The customersare trying to conduct time sensitive experiments, and have now been without a suitable antibody for a week, so its quit urgent for them

ANSWER:

Thanks for sending along the blot image. Here are my thoughts:

1) Where are the markers and marker labels?;

2) Has any other antibody been used successfully with these samples?;

3) It is mentioned 0.5mg protein loaded, Is this the pure MMP7 or colon homogenate?; No more than 0.1mg homogenate should be loaded and 0.5 ug pure protein can be loaded as positive control; loading too much protein will cause proteins to not separate as expected;

4) 15% gel is too high percentage for MMP7 which is 28kDa, can use 12% for better resolution

I hope these comments are helpful. Thanks in advance for the additional information.

Inquiry: Antibody code: ab5706 Batch number: lot:GR1728-7 Antibody storage conditions (temperature/reconstitution etc) When received stored aliquoted and stored immediately at -20 C. Description of the problem (high background, wrong band size, more bands, no band etc.) The antibody seems to be unspecific. Gel shown below shows unspecific binding of the anti-MMP7. Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) Samples included a positive control of purified recombinant MMP7, and samples from DSS colitis induced mouse colon homogenate, with the negative control being healthy mice. Intended purpose was to use the anti-MMP7 for Western Blot and in the future, immunoprecipitation. Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) Sample was prepared in 0.2%BSA. The western blot was incubated in the primary antibody solution, for one experiment overnight, and for the second trial for one hour at room temperature. The incubation time was not the problem, the same result was obtained. Amount of protein loaded: In this gel, ˜0.5 mg of protein was added, which was lowered in the subsequent experiments. Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) Reducing PAGE gel, 15%. Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) Transfer conditions were 200 mA for 1hr 15 min. The transfer buffer was made from 1x glycine buffer and 20% methanol. The buffer was prepared in cold milli-Q water. Two blocking solutions were tried in separate experiments, first 3% milk, 100 mM tris pH 7.5, 150 mM NaCl. The second time, the milk was replaced with BSA. Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) The primary antibody is ab5607. Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) The secondary antibody was goat anti rabbit –HRP from Jackson. Detection method (ECL, ECLPlus etc.) ECL was used for detection. Positive and negative controls used (please specify) The positive control here was purifed MMP7. OPTIMIZATION ATTEMPTS (PROBLEM SOLVING) How many times have you tried the Western? Two separate times, two separate secondary antibodies, two separate batches of ECL (from two different labs). Have you run a "No Primary" control? Do you obtain the same results every time? e.g. are the background bands always in the same place? Yes. Both times the gels showed very unspecific binding.

ANSWER:

Thank you for submitting your completed questionnaire. I am sorry to hear this vial of ab5706 is giving you trouble.

You mentioned including a western blot in your enquiry but unfortunately it did not make it through. If you could, please re send the blot as an attachment.

Thanks! I look forward to hearing from you so that we may resolve this as soon as possible.

Thanks for your kindly reply, after contact this customer, she would like to have a credit note,

could you please help her to create a credit note ?

I deeply appreciate your great assistance!

Best regards

ANSWER:

Thank you for confirming this information and for your help and cooperation with this case.

As requested, I have asked our accounting department to issue you with a credit note. This can then be redeemed against the invoice of a future order.

Credit ID: #####

As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. Please refer to the credit ID number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.