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Inquiry: Dear custumer service, we recently used your HCN4 antibody (ab32675) to perform an immunofluorescence assay on human atrial and ventricular myocytes. Staining was succesful and this data were submitted for publication. One of the reviewer asked to have a negative control, e.g. a cell stained with the HCN4 antibody pre-absorbed with the specific peptide. How can we meet this request? do you have a specific peptide and a suitable protocol to perform this assay? thank you for your assistance. Best
Asked on Jun 18 2012
Thank you for your enquiry and your interest in our products.
The immunogen used to raise this antibody was a synthetic peptide: SHGSLLLPPA SSPPPPQVPQ RRGTPPLTPG RLTQDLKL, corresponding to amino acids 1048-1085 of Human HCN4:SHGSLLLPPASSPPPPQVPQRRGTPPLTPGRLTQDLKL. Unfortunately, the peptide is not commercially available.
Even though the immunogen peptide is not available, you could apply several controls to check the specificity of this antibody:
- isotype control,
- negative control,
- no primary - only secondary control
The isotype of this antibody is IgG1 so you can apply an isotype control using rat IgG1 (ab46745, ab128477, ab18403, ab18404, ab18407).
HCN4 is highly expressed in thalamus, testis and in heart, both in ventricle and atrium. It is detected at much lower levels in amygdala, substantia nigra, cerebellum and hippocampus. Generally, negative controls are cells/tissue which do not express the target protein or at very low level. You may find some useful information about expression level of HCN4 at this site: http://www.proteinatlas.org/ENSG00000138622
I hope this helps and if I can assist further, please do not hesitate to contact me.
Answered on Jun 18 2012