Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Thank you for your prompt answers. Your co-operation in this matter is highly appreciated.
I understand that you have already carried out some optimization steps and it seems that the primary and the secondary antibodies worked on positive control but the signal was very weak on the samples. Is that correct? Would you be so kind to clarify what samples you have been using:
- species,
- cell or tissue types,
- lysate (total cell lysates, or cytoplasmic fraction etc)
I have forwarded your response to our Australian Distributor (Sapphire) to get some confirmation about the shipment and order details. Could you please laise with them to find out the Abcam Order number?
Once again, thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Thank you for enquiry and for taking the time to provide some useful details of the experiments. As I understand three of our products (ab3583, ab13987 and ab97051) have been used in these studies.
Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
- Q1: Could you please confirm the batch numbers of ab13987 and ab97051?
- Q2: Were these products shipped in the same package?
- Q3: Could you please provide the Abcam Order Number(s) or your PON?
- Q4: Have you optimized the dilutions of the primary and the secondary antibody?
- Q5: Is the HRP still active (ab97051)? Have you used it successfully with another primary antibody?
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Thank you for your inquiry.

Please find my answers to your questions below:

1. I recommend to dilute antibodies in PBS or TBS instead of water.

2. The best storage option for antibodies is un-diluted. I suggest to only prepare the amount of diluted antibody that is necessary for one experiment and use it up immediately.

3. If an antibody has to be stored frozen, I strongly recommend to prepare aliquots and only to defrost as much as is needed for one experiment. If the aliquot contains more than that is can be keep at 4C for a couple of days.
As a general guideline, HRP conjugated antibodies should be stored at 4C.
We guarantee our antibodies to work as stated on the datasheet only when stored as instructed on the datasheet.

4. We do have an extensive protocols section on our homepage:

https://www.abcam.com/index.html?pageconfig=popular_protocols

including protocols for

antibody storage:

https://www.abcam.com/index.html?pageconfig=resource&rid=10795

and IHC

https://www.abcam.com/index.html?pageconfig=resource&rid=11384

I hope this information is helpful. Please do not hesitate to contact me again with any further questions.

Thank you for contacting us. I would recommend the following protocol for your purposes:

Fixation

Fix the samples either in ice-cold methanol, acetone (1-10 min) or in 3-4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature.
Wash the samples twice with ice cold PBS
Incubate the samples for 10 min with PBS containing 0.25% Triton X-100.
Wash cells in PBS three times for 5 min.



Blocking and incubation

Incubate cells with 1% BSA in PBST for 30 min to block unspecific binding of the antibodies (alternative blocking solutions are 1% gelatin or 10% serum from the species that the secondary antibody was raised in).
Incubate cells in primary antibody ab97546 diluted 1:100 in 1% BSA in PBST in a humidified chamber for 1 hr at room temperature or overnight at 4°C.
Wash the cells three times in PBS, 5 min each wash.
Incubate cells with the secondary antibody ab97051 diluted 1:500in 1% BSA for 1 hr at room temperature.
Wash the cells three times in PBS, 5 min each wash.



Substrate and mounting

Using kit ab94665, combine30 ul (1 drop)of DAB Chromogen with each 1.5 ml (50 drops) of DAB Substrate.
Apply mixture to cells.
Incubatecells for up to 10 minutes.
Rinsecells in PBS, counterstain with hematoxylin if desired, and mount.



I hope this helps, please let me know if you need any additional information.

Thank you for your inquiry. The MSDS is linked to each online datasheet where the product requires one.  It is on the right hand side of the page under the shipping and pricing information (under the "Datasheet PDF" link). For ab290: https://www.abcam.com/assets/popups/popup_msds.cfm?intMSDSID=3 The secondary ab97051 and protein ab100961 do not require an MSDS as these are non-hazardous products.  Please let me know if you require a statement confirming this. I hope this information helps.  Please contact us with any other questions.

Thanks for your call today and for letting us know about this issue. As we discussed, I'm sending a free of charge vial of ab97051 on the order ***, which should arrive tomorrow. I am sorry for any confusion, and if there is anything else that we can do for you please let me know. Enjoy the rest of your week!

Thank you for your email. Yes, a replacement of ab290 with ab13537 is possible. In order to do so, could you please send me the order details (order number, date, name of distributor) ? Thank you.