Goat Anti-Rabbit IgG H&L (Biotin) (ab6720)

Thank you for your reply.

I would be pleased to provide you with the Abtrial testing discount codes. In order to do so, could you please confirm which distributor you will be purchasing the antibodies from?

I look forward to receiving your reply.

Thank you for contacting us and your interest in our products.

The antibodies you have chosen for your ELISA should work well together. The immunogen used to raise the mouse monoclonal ab54219 does not cover the Y223 region of BTK and they should therefore not be detecting the same epitope.

However, as we have not tested these antibodies together in a sandwich ELISA we would not be able to guarantee their compatibility. For this reason, you would be eligible to take part in our Abtrial testing scheme.

The way this scheme works is that you would purchase ab54219 and ab51210 as normal and test it in your ELISA. As long as you then share the results with us through submitting an Abreview (no matter whether the results are negative or not) you would then receive the value of ab54219 or ab51210 of a subsequent Abcam order.

More information on this offer can be found from the following link:

https://www.abcam.com/abtrail

The only constraint in this offer is that the antibody to be tested must be purchased, the Abreview submitted, and the free credit claimed within a 4 month period.

If you would like to participate in this offer please do let me know as a discount code needs to be issued prior to the purchasing of either ab54219 or ab51210.

You mentioned that you were setting up an Alphascreen. I am sorry but I did not understand how this would work. Are you just going to use one of the primary antibodies for this? If you could explain this a little more I should be able to help further.

I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

Thank you for contacting us and you interest in our products.

There are a few differences between the two pairs of antibodies you have identified. A few of these include the purification used (affitiny vs IgG fraction) as well as the ratio of conjugation. The ab6788 and ab6720 is estimated to have a conjugation ratio of 10-20 per IgG molecule whilst ab97049 and ab97021 have a ratio of 1:4.

I am not familiar with the amplification technique you intend to use and cannot therefore anticipate how these differences may alter the results.

If you could share a more detailed description of the procedure I may be able to advise in more detail.

I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

Thank you for contacting Abcam.

As you mentioned in your email, we at Abcam cannot guarantee that these antibodies would still work after this time, but if have enough samples to run a few test batches to check the validity of the antibodies, then that would definitely be worth trying. If, however your samples are scarce and rather precious, then I would advise playing it safe and purchasing new antibodies.

If there is anything else I can help you with, please let me know.

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. 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 with the order number 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.

Thank you for your message and for providing this further information.

Thank you for taking the time to provide the additional details to me. These have been of great help in understanding the issue. I am sorry to hear that this product had performed unsatisfactorily for you. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or a refund/credit note in compensation.

I look forward to hearing from you with details of how you would like to proceed.

Thank you for contacting us.

I am sorry to hear of the difficulties that you have been experiencing using this product and wish to thank you for performing appropriate steps to determine whether the issue is with the product or protocol.

Would you mind sending me the details of your protocol including the tissue type you are working with and your sample preparations? Any images that you may have and any steps that you have changed while troubleshooting would be extremely helpful as well.
These details will not only help me to better understand your difficulties, they are also used for internal testing of the products if deemed necessary.

Would you also be able to send me the order number for this product?

The antibody is covered under our Abpromise for six months and is guaranteed to work in IHC-P. If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for providing that extra information. It has helped greatly to understand what you have been doing and what may help to improve the signal of the staining. There are a few areas that I would suggest would be worth optimising (if you have not already done so) which may lead to an improved signal. 1. Fixation You have stated that you are fixing the tissue for a minimum of 48 hours. The ideal fixation time will depend on the size of the tissue block and type of tissue but usually 18-24 hours seems ideal for most applications. Over-fixation can lead to the epitope being masked. 2. Antigen retrieval Antigen retrieval can be used to overcome the masking mentioned for the fixation. Depending on the level of masking, different conditions of antigen retrieval may be required. For example, if using a microwave we would usually suggest trying retrieval for 5, 10, 15 and 20 minutes to see which produces the optimal results. 3. Hydrogen peroxide blocking I would suggest performing the hydrogen peroxide blocking following the incubation of the primary antibody if you have not already tried this. The hydrogen peroxide can sometimes affect sensitive epitopes and by performing the step following the incubation with the primary antibody this will not affect the staining. 4. Primary antibody incubation You mention that you have optimised this step by changing the dilution and incubation time. If you have not already done so I would suggest attempting the incubation for 1-1.5 hours at room temperature with agitation. 5. Detection method Different techniques can be used to directly amplify the signal. Currently you are using streptavidin-HRP conjugate which is labelled in a 1:1 ratio. It is possible to employ avidin which has been labelled in a 3:1 ratio with HRP using kits such as Piercenet product 32020. Although avidin can cause greater background compared to using streptavidin. Alternatively HRP polymer can be employed. This consists of using a secondary antibody which is attached to a polymer-HRP complex. Abcam has the following product to offer, ab2891, however this is a little pricey. I would suggest trying to optimise the steps 1-4 and if sufficient progress is not made contemplate employing one of signal amplification methods mentioned. We have quite a detailed guide to IHC which may be of help to you. This outlines many of the different experimental parameters which need to be considered when performing IHC and suggestions to improve experiments: https://www.abcam.com/ps/pdf/protocols/ihc_p.pdf I hope this information has been of help and your staining improves. If you want any further information or help please do not hesitate to contact us again.

Thank you for contacting us. I'm glad to hear you have been getting good staining with ab469. In order to help you obtain more intense staining would you mind filling in the form which I have attached to this email. It will allow me to more fully understand the protocol which you have been performing and any areas in which it may be worthwhile to optimise. Could you also answer the following questions: 1. which HRP conjugate have you been using (streptavidin-HRP? could you tell me the catalogue no. etc) 2. in what dilution have you been using the secondary antibody? ab8114 is supplied at a concentration of 1 mg/mL. The recommended working concentration of this antibody when using it for immunohistochemistry with paraffin embedded sections is 5 µg/ml. This equates to a dilution of 1/200. This is only a guideline and may need to be optimised for the staining which you are performing. I hope this information has been of help and look forward to your reply.

Thank you for your interest in Abcam products. ab2378, ab955 and ab9018 are mouse monoclonal antibodies. I would recommend using ab97044 as a secondary antibody; https://www.abcam.com/Rabbit-polyclonal-Secondary-Antibody-to-Mouse-IgG-H-L-Biotin-ab97044.html ab5690, ab103573, ab10558 are rabbit polyclonal antibodies. I would recommend using ab6720; https://www.abcam.com/Rabbit-IgG-secondary-antibody-H-L-ab6720.html ab53444 is a rat monoclonal antibody. I would recommend using ab6733; https://www.abcam.com/Rat-IgG-secondary-antibody-H-L-ab6733.html To let you know we are running a promotion '4 for 3' on primaries; if you could select 1 more primary antibody then you will be entitled to 8 primary antibodies for the price of 6. Use the discount code and follow the instructions given on website. I hope this information will be helpful. Should you have any other question please do not hesitate to contact me.