Recombinant Dengue Virus 2 NS1 glycoprotein (ab64456)

This protein is purified by affinity chromatography.

On a gel, it can be seen as a monomer, dimer and polymer. We usually recommend heating samples up to 95ºC in SDS-Page before loading and running the gel in reducing conditions.

The molecular weight we observe is around 40-50 kDa.

The ab64456 Dengue Virus NS1 glycoprotein protein was derived from serotype 2, Thailand strain, however it tested positive with all four serotypes.

Although the sequence is proprietary, we can confirm that it contains all domains except the transmembrane domain sequence. This protein also contains 2x6 His tags, one located at the N terminus and the other at the C terminus.

Thank you for your new contact.

As I have explained in my previous e-mail, ab64456 has never been used as immunogen.

Since this glycoprotein is epitope tagged (having additional amino acids ie.HA sequence: YPYDVPDYA) and the storage buffer contains Sodium Azide (cytotoxic agent), Glycerol, Urea, Tris HCl, EDTA, it would not be suitable for applying it as immunogen.

In fact we have some antibodies against Dengue Virus NS1 glycoprotein so the customer would not need to spend and waste time with the antibody production. These antibodies are: ab41616, ab41490, ab41623 and ab138696.

I hope this will be useful for you and your customer.

Thank you for your interest in our products.

The two kits we have for detection of Dengue virus-specific IgG or IgM ab108728 and ab108729, are for detection of antibodies in serological samples that detect Dengue virus. So, they detect an immune response to the virus, rather than the virus itself. On the other hand, antibodies that are raised against the virus, such as antibody ab138696, bind to the virus itself.

For a positive control, I recommend the recombinant NS1 protein, ab64456. A negative control would simply be a blank control, buffer with no protein.

Most of the antibodies you selected have not, to our knowledge, been tested in ELISA of any kind. For sandwich ELISA, I am hoping we can add the detection antibody that complements the capture antibody, ab138696. I expect we will have this on our website in just a few days. I will e-mail you when we do add it.

Like ab138696, it is a mouse monoclonal antibody raised against the NS1 protein. We do not have either antibody in a conjugated format, but we do have conjugation products that make conjugating an antibody simple, which may be necessary, depending on your platform. Enter "Easylink" in the search box on our website to see our selection of conjugation kits. Before conjugation, the antibody will need to be concentrated to the appropriate concentration, at least 1 mg/ml. The product for concentrating an antibody is ab102776, or ab102778 for three units.

https://www.abcam.com/Antibody-Concentration-Kit-1-column-ab102776.html (or use the following: https://www.abcam.com/Antibody-Concentration-Kit-1-column-ab102776.html).

I will contact you when we have the partner for ab138696 up on the website. Please let me know if you have any questions in the meantime.

Thank you for your enquiry regarding ab64456.

It is a Dengue Virus NS1 glycoprotein protein (His tag) which has been tested and characterised for ELISA and WB applications. It has not been used as immunogen to raise an antibody against.

If you need any further assistance in the future, please do not hesitate to contact me.

Thank you for contacting us. I apologize for the delay.

The ab64456 Dengue NS1is from serotype 2, Thailand strain.

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

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My customer would like to know the strain name of Dengue virus protein is from. Could you provide this info?

Many thanks in advance!

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

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

I would like to reassure you that this antibody is tested and covered by our guarantee for WB. Before deciding how to proceed, I would like to investigate this particular case further for you, and also obtain some further information for our quality records to determine what may be the cause of the problem.

In order to do this, I have enclosed a questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily. I have completed the sections for which information has already been provided.

It would also be beneficial if you are able toconfirm:

1. What other samples have been tested with this antibody? Has it detected endogenous protein in any samples? I can suggest it would be beneficial to try this if possible.

2. Has the protein been stained with comassie on an SDS gel? Is it intact and running at the correct molecular weight?

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.

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


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.

Gracias por tu respuesta.
He consultado esta información con mis compañeros del laboratorio.
La glicoproteína (ab64456) contiene toda la secuencia excepto el dominio transmembranal. Además contiene 2x6 His tags, uno localizado en el extremo N terminal y el otro en el C terminal.
Respecto al anticuerpo (ab41616) me comentan que el dímero del virus Dengue NS1 está presente en las muestras sin calentar (forma sin reducir), y por efecto del calor se convierte al monómero. El epítopo es por tanto conformacional. En cualquier caso imagino que para llevar a cabo el ensayo ELISA no usáis muestras reducidas. Si tenéis la oportunidad os aconsejaría que probarais la combinación de proteína /anticuerpo mediante WB usando las muestras sin reducir, para comprobar si el anticuerpo realmente está detectando la proteína.
El epítopo del anticuerpo no se ha mapeado, por tanto no puedo saber si reconoce el extremo N terminal o C terminal de la proteína donde se localizan los His tags.
En cualquier caso, como no hay más Dengue Virus NS1 glicoproteínas en el catálogo, puedo mandaros de reemplazo otro primario contra la misma, esperando que esta vez si la reconozca. Estas son las alternativas:
https://www.abcam.com/Dengue-Virus-NS1-glycoprotein-antibody-DN1-ab41490.html
https://www.abcam.com/Dengue-Virus-NS1-glycoprotein-antibody-DN2-ab41623.html]
Espero vuestra respuesta para saber cómo proceder y qué solución os podemos dar.

Thank you for contacting us.
I am sorry to hear you are having problems with one of our products. I appreciate the details provided. I am afraid that product ab41616 is not guaranteed to work in ELISA. We have tested this antibody in WB and ICC, but we are not aware of its suitability for ELISA assays.
As far as I understand you are performing an indirect ELISA, coating your plate with the antigen ab64456 and then capturing it with the monoclonal antibody, using ab20043 (secondary) to detect your primary, is that correct? It could well be the primary antibody is not recognising ab64456 as it may not work for this application.
I can also confirm the peptide ab64456 is tagged with 6 histidines. It is also possible the antibody is not detecting this protein if the epitope it recognizes is masked with the histidine tags.
If you wish to I can try to find out the epitope targeted by ab41616 and check whether it is comprised within the tag region.
Regarding the failure of ab1187 (Anti-6X His tag® antibody (HRP)) to detect the glycoprotein, it could be due to the presence of sodium azide to preserve the protein. The antibody is linked to HRP and this enzyme is very sensitive to sodium azide.
In any case, as all of our products are covered by our Abpromise guarantee, if you wish to receive a replacement for ab64456, I will be more than happy to send it to you.
However, if you want to receive the same protein, you may still experience the same problems. In order to target this protein, I can try to find within our catalogue a primary against it that does not detect the epitope within the tagged sequence, or any other solution you may prefer.
I look forward to receiving your reply.

I am sorry for this misunderstanding.

We do not know which protein ab9200 exactly recognises and therefore I suspect this antibody will not be suitable for your purposes.

We onlyhave a mouse monoclonalantibody against Dengue Virus NS1 glycoprotein antibody that recognises all serotypes:

ab41616

https://www.abcam.com/Dengue-Virus-NS1-glycoprotein-antibody-DN3-ab41616.html (or use the following: https://www.abcam.com/Dengue-Virus-NS1-glycoprotein-antibody-DN3-ab41616.html).

I am sorry that we do not have a product that fits all your specifications.

May I suggest to try the Biocompare website which has a excellent antibody search facility:

http://www.biocompare.com/

I apologise that I could not be more helpful on this occasion and wish you good luck with your experiments.