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I purchased Anti-HTLV p19 Antibody [TP-7] (ab9080) Lot GR67109-1 to use
to stain HTLV-1 infected cells by immunofluoresence.
This clone of p19 antibody was used by Pais-Correia et al 2010 published
in Nature Vol 16, No 1, pp83-90 for staining HTLV-1 infected cells. The
authors noted no staining on non-infected cells. However, the antibody
appeared to stain non-infected cells and also stain cells when they
werent permeabilised, which is not expected. It appears the Ab is
staining non-specifically the plasma membrane of infected but also
non-infected cells thus invalidating the Abs specificity.
I wonder if you could help me with this problem or offer a replacement
The protocol i followed was roughly outlined in the paper mentioned
above and used the concentration outlined on the Abcam product
datasheet. The protocol:
Coat glass coverslips with poly-lysine for 30mins then wash with water
and leave to dry
Fix HTLV-1 infected MT2 cells or non-infected CEM cells in 3% PFA for
20mins at room temp. Wash off PFA with PBS containing 50mM NH4Cl and
incubate in this solution 20mins to quench free aldehydes.
Prepare permeabilisation or intact staining solutions: PBS + 1% FBS +/-
0.05 % saponin.
Incubate cells in staining buffer for 30mins for blocking +/-
Replace with primary antibody = Anti-HTLV p19 Antibody [TP-7] (ab9080)
at 10µg/ml (stock solution is 1.4mg/ml diluted 1:140) or the same
concentration of isotype control IgG1
Incubate 1hr room temp
Wash x3 in staining buffer (intact or perm)
Add on alexa fluor 488 goat anti-mouse IgG (H+L) from another company diluted 1:500 with staining buffer (perm or intact)
Incubate 1hr room temp
Wash x3 in staining buffer
Wash x2 in PBS alone
Incubate cells with DAPI
Wash off DAPI, mount cells.
Please see attached images for representative staining observed which
highlights the issue that the Ab is staining unexpectedly non-infected
cells and also intact cells.
Asked on Feb 23 2012
Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.
Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.
I apologize for the inconvenience and I'd be happyto offer you a free of charge replacement, credit note, or refund in compensation. The free of charge replacement can be arranged with another antibody to the same target (e.g. ab9079) or an antibody to a different target.
Please let me know which option would be best for you.
I look forward to hearing back from you and resolve this case for you.
Answered on Feb 23 2012