Anti-Fibrillarin antibody [38F3] - Nucleolar Marker (ab4566)

I am using confocal microscopy with hippocampal neurons. I see the appropriate staining in the nucleolus but also a fair amount of staining in the cytosol. I have used no primary controls and it is not a problem from any non-specific secondary staining. Is there any advice you can give me, or is this known to happen at all in neurons for this target?

ANSWER:

Since you can see expected positivity along with what appears to be non specifici cytosolic staining, I would suggest diluting the primary 2 - 4 fold, which will hopefully reduce non specific background while retaining specificity.

Also, if you are not already, I recommend overnight incubation of the primary at 4 degrees C.

I hope this is helpful. Please contact us again if you have any further questions.

DESCRIPTION OF THE PROBLEM No signal or weak signal
SAMPLE Cell extract MCF7 Human breast cancer cell line
PRIMARY ANTIBODY AB4566
DETECTION METHOD ECL
POSITIVE AND NEGATIVE CONTROLS USED Trying to use this ab as +ve contro. Nuclear fraction +ve for CTCF
ANTIBODY STORAGE CONDITIONS 4C as supplied
SAMPLE PREPARATION Nuclear extracts, proteas inhibitors samples heated 90C
AMOUNT OF PROTEIN LOADED 5mg
ELECTROPHORESIS/GEL CONDITIONS 4-12% Bistris SDS gel + DTT in sample buffer
TRANSFER AND BLOCKING CONDITIONS iBlot (invitrogen) to PVDF membrane, blocked 5% milk in PBST 60 minutes
SECONDARY ANTIBODY D alpha M HRP Conj 1:5000
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1
HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? Tried this after also getting no signal with IF in this antibody

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results for your customer.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab4566.


How long has the antibody been stored at 4C? We recommendupon delivery aliquot and store at -20C or -80C.


Did the customer specify his/her nuclear fractionation protocol? This is what we would recommend: http://www.abcam.com/index.html?pageconfig=resource&rid=11473


How much protein is the customer loading on the gel? I can't imagine he/she is loading 5 mg. We generally recommend loading 20ug-30ug.

Also how long did they heat at 90C?


Is the customer seeing any bands at all? Have they done a ponceau stain to confirm the efficiency of the transfer? I see that they saw positive results with CTCF. Was that on the same membrane?


At what concentration/dilution is the customer using the primary antibody? We would recommend 1/500 overnight at 4C.


Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

 DESCRIPTION OF THE PROBLEM No signal SAMPLE MCF7, HELA Human Cell lines PRIMARY ANTIBODY AB4566 DETECTION METHOD Fluoresence Microscop - Deltavision, DFC500 lcica POSITIVE AND NEGATIVE CONTROLS USED No primary, IgG-ve nucleolin +ve Dapi - nuclei ANTIBODY STORAGE CONDITIONS 4C as supplied FIXATION OF SAMPLE 4% Paraformaldehyde, 10 min RT ANTIGEN RETRIEVAL N/A PERMEABILIZATION STEP 0.2% Triton X-100 in PBS, 10 min RT BLOCKING CONDITIONS 20% Blocker in PBS + 0.02% Triton X-100 or 0.1% Triton X-100. 7.5% NGS SECONDARY ANTIBODY Invitrogen alpha M AI4888 1:500 1hr HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes D O YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Ab concentration, blocking ADDITIONAL NOTES Customer also tested in WB (enquiry coming)

ANSWER:

Thank you for contacting us.

I am sorry to hear of your customer's problems with this product. Could you please double check the purchase order provided as there does not seem to be an ab4566 associated with this PO.

Could you provide me with the antibody concentrations used as well? I would also suggest trying a methanol or acetone fixation without a permeablization step as paraformaldehyde fixation may mask the epitope sites.

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

Thanks a lot for your prompt reply. I actually used three different dilutions: 1:250, 1:500, and 1:1000 but none gave me nucleolus staining. I can only see diffused dots thoughout the cytoplasm and nucleoplasm.

  I really appreciate if you kindly give me a nucleolus marker antibody replacement. Either Nucleolin antibody (ab22758) or mouse fibrillarin antibody 38F3 should be great, as long as they are certified in immunofluorescence.

  Again, thank you very much for your generous help!  

ANSWER:

As requested, I have issued a free of charge replacement, ab4566, mouse fibrillarin antibody 38F3.

To check the status of the order please contact our Customer Service team and reference this number. You should expect the antibody on Tuesday of next week.

There is a specific cell fixation protocol for staining cells with this antibody. Your protocol (fixation with PFA for 30 minutes followed by permeabilization) may also work, but this was the protocol used to produce the three large images at the bottom of the online datasheet. In brief, cells are fixed with formalin at room temperature for only one minute, then further fixed with cold methanol for only one minute. The protocol is at the following link:

http://www.abcam.com/assets/popups/popup_datasheet_protocols_printer_friendly.cfm?pid=179&intAbId=4566

The online datasheet is here:

Click here (or use the following: http://www.abcam.com/index.html?datasheet=4566).

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

I was looking for an antibody that would be a nucleolar marker in plants. as I would like to try it in sections that will contain several cell layers, I'm not convinced that the antibody will work well. Is there any possibility of you sending me a sample trial, just a tiny bit, to see if it may works. Only after trying it, I would like to take the order decision . I would be very happy if you could send me a trial sample.

ANSWER:

Thank you for your enquiry. Ab4566 does react with Plants and has been successfully tested for application in Western blot and Immunocytochemistry/Immunofluorescence.

We do not routinely offer free or trial sized samples for testing purposes. 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 Abpoints which can be redeemed on a number of rewards (a further 100 Abpoints will be offered for an image).

Please contact us again if you have any additional questions.