Anti-ACADL/LCAD antibody [7F5DD6] (ab128566)

I am sorry to hear that you have been experiencing problems using this product in the application that you wish.

In order to assess the quality of our products I would ask that you complete a brief questionnaire relating to the application used. Often it is possible to make suggestions that may help resolve problems experienced using a particular product.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience.

Could you provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

Thank you for providing that information.

As discussed over the phone, the lab have provided the Western blotting protocol they use with this antibody. Please find it in the attachment to this email.

From having reviewed your protocol, there are no great differences in how you have performed yours compared to ours. However there are a few things I would suggest changing in order to optimise the results observed:

1. I would recommend using a lysis buffer such as RIPA, taking care to keep the samples on ice throughout the lysis process. This should lead to a greater proportion of the protein being extracted and reduce any degradation. It is also important to include protease inhibitors in your lysis buffers to reduce the level of degradation of the proteins. The high, non-specific background observed could be due to breakdown products being detected.

2. I would also decrease the amount of antibody used. We recommend using about 1 µg/ml. This equates to a dilution of 1/950. This reduction in the amount of antibody used could also reduce the background observed.

We have a more detailed explanation of this in the following protocol book:

https://docs.abcam.com/pdf/misc/abcam-protocols-book-2010.pdf

I also have a few further questions if you wouldn't mind:

1. Has the secondary antibody been used previously with any other antibodies? What was the result?

2. You described the secondary antibody as reacting with mouse and rat, could you explain this? Could you provide the catalogue number and manufacturer of the secondary antibody?

3. Was any loading control used with the samples prepared such as tubulin or VDAC1/Porin? This can be very useful to verify that the transfer has proceeded as expected as well as ascertain the quality of the samples.

4. Could you explain what the different blots shared are? The one on the left and right? Could you label what has been loaded in each well as well as mark the approximate molecular markers?

5. What exposure time was used for each blot?

With this information I may be able to provide a few further suggestions to hopefully improve the results observed so far.

I look forward to receiving your reply.

Thank you for taking the time to complete our questionaire. The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab128566. I would also appreciate if you can confirm some further details:

1. Please confirm which species was the sample from? Was this sural nerve tissue or a cell line of some type?

2. Reviewing the image originally provided, there is a lot of general background on the blot. This can indicate there is crossreactivity of the antibody with the blocknig agent. Therefore, I can recommend to try BSA rather than milk to block. Changing blocking agent can often help to improve the results.

3. We recommend to load 20 - 30 ug per lane of the gel. This will ensure there is enough protein for detection.

4. How was the sample prepared? I can suggest to use RIPA lysis buffer which should provide a suitable protein preparation. Then sample buffer containnig reducing and denaturing agents can be added before heating.

5. Could you confirmi the wash steps that have been used? I can suggest to try PBS contiaiing 0.2% Tween. Wash 4 times for 5 minutes at ech appropriate wash step.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Thank you for calling us and for alerting us to the problem you are experiencing with the Anti-ACADL antibody [7F5DD6] (ab128566). We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

As we discussed on the phone, I have requested the protocol used in Western blotting and will share this with you once I have it. In the meantime, if you could complete the questionnaire I have attached to this email I can have a look to see if there is anything I can suggest may be contributing to the problems encountered.

This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

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

As requested, I have contacted our Mitosciences laboratories in order to obtain the western blot testing protocol for you.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for WB and mouse and rat samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

While we are waiting for the protocol, I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I am sorry the image provided was not in a format that I can access on our system. I would appreciate if you couldtry sending an image again as an attachment in JPEG format. This would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.



Order Details
Antibody code:

Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.