Anti-Nanog antibody - ChIP Grade (ab21624)

Hola,
Hemos seguido vuestras sugerencias y hemos obtenido los mismos resultados. La membrana se vela completamente cuando utilizamos este anticuerpo.
No dudéis en contactar con nosotros si necesitais más información.
Un saludo,

ANSWER:

Gracias por tu respuesta.

Lamento mucho que no hayáis conseguido mejorar los resultados obtenidos. Dado que el anticuerpo está en garantía, si lo deseáis puedo enviaros un reemplazo del mismo anticuerpo (aunque el único lote en stock es el mismo que os hemos enviado esta última vez) o un nuevo anticuerpo contra la misma proteína. Hay numerosas opciones, te mando el link al resultado de la búsqueda para que las consideréis.

http://www.abcam.com/index.html?pageconfig=searchresults&search=Nanog&pt=1&sk=app&sv=69&sn=WB&l=2&fViewMore=1

Si lo preferís puedo enviaros en su lugar un reembolso o vale sin fecha de caducidad para futuras compras en Abcam.

Quedo a la espera de tu respuesta para que me indiques cómo proceder.

Hola,
Adjuntamos los formularios con los detalles solicitados,
Por favor no duden en contacar con nosotros si necesitan más información,
Un saludo

ANSWER:

Muchas gracias por enviarnos el cuestionario relleno y las imágenes de los blots. Lamento muchísimo que el anticuerpo no os haya dado buenos resultados, y después de haber estudiado el protocolo me gustaría haceros algunas sugerencias y preguntas para intentar mejorarlos.

No acabo de entender muy bien la imagen enviada, ¿sería posible ver una imagen completa de la membrana, con los marcadores de peso molecular, y la información acerca del contenido de cada calle? ¿A que corresponden los distintos experimentos?

-Dado que la proteína se expresa a nivel nuclear, un lisado de la fracción nuclear daría probablemente resultados más limpios e intensos correspondientes a la proteína de interés.

-Reducir la carga de proteína suele ser eficaz para reducir también el fondo.

-Para este anticuerpo hemos comprobado que 1%BSA como agente de bloqueo genera una señal más limpia que la leche, y la dilución óptima de trabajo del mismo está entre 1/300-1/500.

En cualquier caso, al estar al anticuerpo bajo garantía (por haberse comprado en los últimos 6 meses), en caso de que las sugerencias no consiguieran optimizar el protocolo, estaré encantada de ofreceros un reemplazo del mismo, o un vale sin caducidad si preferís.

Quedo a la espera de su respuesta. Que pase una buena tarde.

See attached troubleshooting file.

ANSWER:

Thank you for contacting us. I am sorry that this antibody has been giving no signal in WB. I would like to ask you a few questions about your protocol and offer a few suggestions for improving your results:
1) What type of mouse cells were you testing? What was the cell line or tissue type? Were the samples reduced and boiled prior to loading?
2) Could you please further explain what the different lanes in your blot represent? Was this an IP experiment? If so, could you please specify which antibodies were used for pull down, and which were used for blotting?
3) For western blotting, we have found that using 1% BSA as a blocking agent gives a stronger signal that milk. This antibody may also work better at a higher concentration (1:300 - 1:500) with an overnight incubation at 4C.
I hope this helps, if not, please let me know and I will be happy to assist you further.

I have a customer who wants some trial samples. We met him at a conference. Is it possible to help him out. Please refer to his e-mail:

We are interested in antibodies to histone modifications and stem cells. There are specific antibodies in your catalogue that we would like to test out. Regarding the histones: ab8898 and ab4441are yet to be confirmed in cow, ab1186 has yet to be confirmed in cow and in IP application, and ab662 and ab4729 are both untested in IP. We would be keen to evaluate each of these unknowns as each of these antibodies could be very useful for the studies we have begun. The histones that each of these antibodies detects have been identified in preliminary mass spec. studies we are currently carrying out in bovine cells and tissues. Additionally, we are keen to evaluate your antibody to HP1alpha (ab9057) for bovine application.

With regard to stem cell markers, we are keen to find antibodies that work in bovine stem cells for sox2 (ab15830), nanog (ab21603 and/or ab21624) and oct4 (ab18976). We could test each of these using established applications such as IF and/or WB. I hope this gives you a clearer indication of our interests and the applications we wish to pursue, and clarifies why we would welcome the chance to evaluate trial samples of each.

Thank you for your consideration and if we can provide any further information I would be pleased to do so.

Please feel free to contact me if you would like any other information

Kind regards

ANSWER:

Thank you for your enquiry.

Free or trial sized samples are considered on a case by case basis. On this occasion unfortunately I cannot offer you a free sample. Our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement.

Should you decide to go ahead and purchase this product, please let us know how you get on by submitting an Abreview and in return we will offer you 50 Abcam Points which can be redeemed on a number of rewards (a further 100 points will be offered for an image).

Please do not hesitate to contact me should ou require further assistance.

What is the homology of your abs to bovine nanog (DQ069776)?

ANSWER:

Thank you for your enquiry and your patience in waiting for my reply.

I have compared the immunogen sequences with the sequence of bovine nanog using a ClustalW alignment. For ab14959 less than 30% of the sequence is homologous, for ab21624 the homology is 50%, and for ab21603 the epitope is not known but the overall sequence homology is approximately 50%. The end result in my opinion is that the homology is not high enough for any of these antibodies to cross-react with bovine nanog. You would want to see a higher degree of homology to have any confidence that these antibodies would cross-react with bovine nanog.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information,