Anti-Rotavirus抗体(ab20036)

I have been in contact with the lab regarding the issues which you have been experiencing with ab20036. Per the lab, this product does stain the cytoplasma of infected cells, but it is not a strong antibody in IFA. The lab recommends using it at a 1:100 to 1:200 dilution. They also recommend fixing the cells with cold acetone as the 10% formalin may destroy some of the epitopes.

This is a polyclonal antibody that was raised using the whole virus, so it reacts with a number of the viral proteins. In ICC/IF, it gives cytoplasmic staining.

Thank you for taking the time to contact us. I am sorry to hear you have some concerns regarding the results from this antibody.

I can confirm the immunogen used to producethis antibodyis Rotavirus NCDV (Nebraska Calf Diarrhea Virus). I'msorry wedo not have the amino acid sequence available for the complete virus.


I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for immunofluorescence and conjugation. 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.

I am sorry to confirm this antibody is regrettably not tested and covered by our guaranteed in western blot. Could you confirm if it has been tried in ICC-IF which is covered by our guarantee.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily. I have filled in the information already provided.

I would appreciate if you could also provide an image which would help us to assess the results.

After reviewing the details, there are somerecommendations I can suggestto consider:

1. I can suggest to use a lysis buffer such as RIPA to prepare the sample. This should provide a more suitable protein preparation.

2. Has the transfer to the membrane and quality of the sample been assessed with a loading control?

3, Has the amount of virus in the sample been assessed by any other methods?

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



Order Details
Antibody code: ab20036

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.)
PBS buffer with 0.02% Tween 20 for extraction including Sigma proteinase inhibitor cocktail.

Amount of protein loaded
loading 25-35 ug protein per lane

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
12% SDS-PAGE then transfer to PVDF membrane


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Transfer 11 Volts, 2 hours

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
1:5000 dilution of ab20036 (4.5 mg/ml overnight incubation @ room temperature) followed by thrice 10 min washes with PBS-T

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
ab97120 (GR62374-2, donkey anti-Goat IgG HRP, 1:2500, 0.5 mg/ml, 2 hour incubation @ room temperature) and once more thrice 10 min washes with PBS-T before ECL detection

Detection method (ECL, ECLPlus etc.)
ECL

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.