Anti-HP1 alpha antibody - Heterochromatin marker (ab9057)

Could you kindly let me know the isotype for the following antibody: http://www.abcam.com/HP1-alpha-antibody-Heterochromatin-marker-ab9057.html It is listed on that page as "n/a"  

ANSWER:

Thank you for contacting us.

The isotype of this rabbit polyclonal antibody is IgG. I have updated the datasheet accordingly. Thank you for pointing this out to us.

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 your suggestion. After try ABCAM protocol and change fixative to ice cold-acetone (for 5 or 10 min) or ethanol. The picture is worst than obtained from our protocol and more non specific and also not bind in heterochromatin region. I will try to stain in human cell and other company HP1 alpha antibody, and will send the picture to you soon.

Regards

ANSWER:

Thank you for getting back to me.

I appreciate the steps that you have taken to optimise this antibody. I am disappointed that there was not a significant improvement in the results that you obtained.

I look forward to receiving your results from the application of this antibody to a human cell line.

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

DESCRIPTION OF THE PROBLEM non-specific staining, and non-heterochromatin marker in mouse

SAMPLE mouse fibroblast

PRIMARY ANTIBODY Rabbit antiHP1alpha, 1:200 incubate overnight at 4 C.

DETECTION METHOD fluorescent microscope

ANTIBODY STORAGE CONDITIONS -20 C

FIXATION OF SAMPLE 4% Paraformaldehyde

PERMEABILIZATION STEP 0.1-0.4 % Triton X-100 30 minute

BLOCKING CONDITIONS 2% BSA

SECONDARY ANTIBODY Donkey antirabbit conjugated with FITC, 1 h in room temp.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Please note in your product sheet, this antibory does't work in mouse species. Stop try to convince people to belive it may work by your prediction, and please refund to us.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. I appreciate the image that you have sent me in order to support your enquiry. Rabbit polyclonal to HP1 alpha has been applied and is guaranteed for its application to human cells. However, customer feedback has suggested that this antibody does not generate the correct staining patterns in plant cells. Whilst we make the prediction that this antibody works in mice, due to 100 percent sequence similarity we cannot guarantee it for this purpose until we receive results that support this from our customers or from our in house testing. It is currently in the shortlist for internal testing and will be tested shortly.

I have read your technical questionnaire and I would like to make a few recommendations.

I would like to recommend you try fixing your cells using ice cold-acetone (for 5 or 10 min) or ethanol. I would also like to suggest that you try incorporating a no primary control experiment in order to ascertain that the result ubiquitous staining that you are observing is not attributable to the secondary antibody.

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

BATCH NUMBER 100191 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM I got a wrong band. It was a bigger than predicted and I used Hela cell lysate for a positive control. But I couldn't get a correct band.

SAMPLE Human fibroblast cell extract and Hela cell extract, HA-tagged HP1?

PRIMARY ANTIBODY 1/500, 1/1000 (with milk, without milk) RT, 1 h or 4? O/N 1X PBST buffer, 3 min X 3

DETECTION METHOD ECL solution (Roche)

POSITIVE AND NEGATIVE CONTROLS USED cell extraction transfected with HA-tagged HP1

ANTIBODY STORAGE CONDITIONS -20?

SAMPLE PREPARATION I used the RIPA buffer to prepare our protein.

AMOUNT OF PROTEIN LOADED 30 ug

ELECTROPHORESIS/GEL CONDITIONS 15% SDS-PAGE or 12% SDS-PAGE

TRANSFER AND BLOCKING CONDITIONS Transfer buffer (25mM Tris, 192mM Glysine, 20% metanhol) 100 V, 70 min, 5% skim milk (in 1X PBST)

SECONDARY ANTIBODY Amersham, anti-rabbit IgG, peroxidase, 1/5000, RT, 1 h 1X PBST buffer, 3 min X 3

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I've altered cell type and antibody binding condition to get a better data

ADDITIONAL NOTES I couldn't get a good data eventhough I used cell extraction transfected with HA-tagged HP1 and Hela cell lysate you recommened for positive control

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. HP1 alpha antibody - Heterochromatin marker (ab9057) is a good selling antibody that we produced in house. I would very much appreciate it if you could provide me with details of the molecular weight that you have been detecting in addition to a representative blot. We often find that commercially available molecular weight standards can very significantly from one to another; often providing the user with an artificially high or low molecular weight. Can you also please confirm that you have detected other proteins with accuracy within this molecular weight range?

advise on this HP1a antibody. Product code is ab9057. I intend to use it for Western blotting. Can I find out whether this antibody is to be diluted in 1% milk or in BSA?

Thanks.

ANSWER:

We recommend that you block the membrane in 5% w/v BSA (fractionV) NOT MILK (milk contains casein which is a phosphoprotein; This is why it causes high background because the phospho-specific antibody detects the casein present in the milk). If you need any futher information, please son't hesitate to contact us. http://www.abcam.com/assets/pdf/protocols/Western%20blotting%20of%20phospho-proteins%20protocol.pdf