Recombinant Anti-Hemoglobin subunit alpha antibody [EPR3608] (ab92492)

I have processed your request to receive ab109444 as a replacement for ab92492. I have also requested that you receive a new vial of secondary antibody and a new HRP substrate kit as well. I do not have an estimated time of arrival yet, but I will keep you updated when I find out more information.

Vielen Dank für Ihren Anruf.

Es tut mir leid, dass Sie Probleme mit diesem Antikörper hatten. Wie am Telefon besprochen habe ich eine kostenlose Ersatzlieferung für Sie in Auftrag gegeben. Sie hat die Referenznummer 123456und sollteMorgen bei Ihnen ankommen.

Ich wünsche Ihnen viel Erfolg für Ihr Projekt. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Thank you for contacting us. I am sorry to hear that ab92492 is producing unsatisfactory results in WB. All of our Hemoglobin subunit alpha antibodies have been validated for use on endogenous proteins, but have not been tested on purified hemoglobin protein. In response to your questions:

1.) This was performed as a reduced and denatured WB using a Laemmli Sample Buffer containing SDS and beta-mercaptoethanol.

2.) The antibody was validated using 5% milk for blocking and 5% BSA for the antibody diluent. We do not currently have any feedback to suggest that this antibody performs better with one blocking agent over another.

3.) The wash and dilution buffers were TBST containing 0.1% Tween-20.

4.) We would expect this antibody to produce good results using any standard WB protocol, but if you could send me the details of your protocol and a description of the problem (no signal, high background, etc), I will be happy to make more specific recommendations to help optimize your results.

5.) The testing was performed using a 1:1000 dilution for the primary antibody with a 1:2000 dilution of the secondary antibody. Rabbit monoclonal antibodies are well known for having very high binding affinities, however, so it is possible that the antibody may be suitable for use at a lower concentration.

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

Thank you for sending that protocol information.

If I have read the information correctly, then there are a few protocol variations that may work:

1 - Did the customer only try a 1/5000 primary antibody dilution? If so, I strongly suggest that they try range of dilutions from 1/500-1/2000, to see if they can find the concentration that will work for them.

2 - According to the protocol, they loaded 5ug of protein on the gel. Did they try increasing the amount of protein, to say 20ug?

3 - How long did they incubate the primary antibody for? We recommend overnight at 4C.

4 - Did they try using 5% milk as a blocking reagent instead of BSA?

If the customer has not tried the protocol variations above, then I would strongly suggest that they do. If they have already tried those variations, then I would be happy to process a refund for this antibody, as it is not working as we guarantee.

I look forward to your reply.

Thank you for your interest in Abcam. We continually add antibodies and other protein detection products to our catalogue but we do not offer custom-made antibodies. However, we are happy to pass on the contact details of the following companies that do offer this service: United States Pocono Rabbit Farm and Laboratory (PRF&L) PRF&L PO Box 240 Canadensis, PA 18325 Tel: 800-622-6381 - Toll Free in the United States & Canada Tel: 570-595-7108 - Worldwide Fax: 570-595-9277 Website: www.prfal.com Maine Biotechnology Services, Inc. 1037R Forest Ave. Portland Maine 04103 Tel: 800-925-9476 or 207-797-5454 Fax: 207-797-5595 Website: www.mainebiotechnology.com Cocalico Biologicals, Inc. PO Box 265 449 Stevens Road Reamstown, PA 17567-0265 Tel: 717-336-1990 Fax: 717-336-1993 Website: www.cocalicobiologicals.com United Kingdom Charles River Laboratories International, Inc. Tel: +44 (0) 1843 823575 Website: www.criver.com I hope this information is helpful to you. Please do not hesitate to contact us if you have any questions.  

I have talked to my colleagues and I will be able to send a new secondary antibody aswell as a new ECL kit with the replacement antibody. I will send the new products as soon as*****decides which antibody he would like to receive, either ab109444 or ab102758.

Gracias por contactarnos de nuevo y enviarnos más información acerca del protocolo seguido. -¿Habéis llevado a cabo un control no primario? Esto es, incubar directamente el secundario y observar si existe tinción sin necesidad de haber añadido el primario. -Mencionas que el secundario es Rabbit biotinilado ¿quieres decir que la especie de la que procede el secundario es rabbit? ¿Contra qué especie está dirigido? ¿Habéis probado otras diluciones del secundario? Quizás obtendríais mejores resultados con diluciones menos concentradas. -Para evitar la tinción inespecífica y con mucho background que apuntáis, es imprescindible bloquear la biotina endógena del tejido, pues es la principal fuente de señal de fondo. -Nosotros normalmente llevamos a cabo el proceso de recuperación de la proteína a diferentes tiempos (5, 10, 15, 20, 25 y 30 minutos). Es posible que el método utilizado sea demasiado agresivo, lo cual puede haber afectado a la morfología de la sección, dando lugar a una tinción inespecífica. Si pudierais enviarnos una imagen de cualquiera de los tejidos teñidos con el anticuerpo, nos ayudaría mucho a entender mejor el problema y facilitaros soluciones. En caso de que estas sugerencias no ayuden a obtener mejores resultados, por favor no dudéis en contactarnos de nuevo, estaremos encantados de seguir ayudándoos.