Anti-MMP9 antibody [56-2A4] (ab58803)

Please update me if you have answers to my questions.

ANSWER:

Thanks for your email.
I apologize for the delay. I am currently awaiting a reply from the lab that supplies us ab58803. I have contacted them again just now and encouraged them to reply as soon as possible.
Thanks for your patience. I will forward you any information as soon as I receive it.

Dear technical support:
This customer raised an inquiry about ab58803 (Anti-MMP9 antibody [56-2A4]), and this customer used this antibody conduct the western blotassay with rabbit sample, and the data shows the multiple bean and high background, because this antibody is already out of our warranty period, therefore she only wants to ask for your kindly help to offer any suggestion to her. I attach the western blot data and the WB questionnaire in this letter, could you please help this customer to solve this problem.
Thanks for your kindly assistance.

Best regards

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I have a few suggestions which may help to improve the results observed.

1. This antibody is able to detect both the 92 kDa latent and the ˜83 kDa active forms of MMP9. This may be the two main bands that you have been detecting in your sample. The band might not migrate to exactly 92 kDa, this is not unusual. Themigration can be shifted slightly upwards or downwards of the expected size due to differences in the loading buffer and protein sample. In order to observe the bandsmore clearly I would firstly suggest to reduce the amount of protein loaded on the gel (I would suggest initially using a 1/10 dilution of your serum).

2. I would suggestheating your sample (heating to 70°C for ten minutes) prior to loading the protein onto the gel as well as including a reducing agent such as DTT or b-mecaptoethanol in the loading buffer if you have not already done so. This will help do denature the protein and reduce the disulfide bonds.

3. I would try switching to 3% BSA in TBST or PBST for the blocking of the membrane. This can sometimes have a dramatic effect on the non-specificity observed. I would also include 1% BSA in the primary and secondary antibody diluents (along with 0.1% Tween again).

I hope these suggestionsimprove the resultsobserved,if they do not pleasedo let me know.

for ab76003 and ab58803 MMP antibodies I want to know if you have pictures or data about the use fo these products with rat tissue in IHC-P experiments thanks claudia

ANSWER:

Thank you for your enquiry.
Typically all the data we have regarding use of our antibodies is posted on the online product datasheet. This includes images from internal testing and user-submitted data (Abreviews) as well as citations in which the antibody has been used.
I checked the datasheets for ab76003 and at this time we do not have any IHC-P data for rat tissues.
It does appear a reference which used ab58803 shows IHC-P data for rat but I am unable to access that article. Here is the citation so you can check the reference:
Hayashi T et al. Quantitative analyses of matrix metalloproteinase activity after traumatic brain injury in adult rats. Brain Res 1280:172-7 (2009). IHC-P; Rat. PubMed: 19464272
I hope this is helpful. Please contact me again if you have any further questions.

ab58803 gave no bands in WB using positive control ab3871
ab39973 gave no bands in WB using positive control ab29466

ANSWER:

Thank you for contacting us.

As we discussed on the phone, you may be able to improve your WB results by switching from milk to BSA and by using a higher concentration of primary antibody (1:500 for example). If you are unable to improve your results, please let me know and I will be happy to offer you either replacements with alternative antibodies or credits on your original purchase. Please let me know how you would like to proceed and I will be happy to assist you further.

I am working with ab58803 and the positive control lysate ab3871. I am not able to see a band for MMP9 in either my sample (pig heart) or the positive control lysate. However, I am not sure if my sample is supposed to express the protein. Should I determine first if the lysate is functioning correctly? Should I test it for beta-actin?

ANSWER:

Thank you for contacting Abcam regarding ab58803.

I am sorry that you have been experiencing difficulties with this antibody in WB. As you discussed with my colleague on the phone, I look forward to hearing back from you after you have tested the lysate wtih beta-actin to confirm that it is not degraded. Please do not hesitate to contact me if you have any additional questions or concerns.