Anti-CFTR antibody (ab2916)

I'm very sorry to hear one of your customers is experiencing problems with ab2916, indeed CFTR is known to be present in sperm and should be detectable with ab2916. I think the problem comes from the fixation which is damaging the epitopes. I would recommend to fix the cells in methanol for 5 minutes at -20C. I enclose below the protocol used by Walker et al (Walker J et al. Production and characterisation of monoclonal and polyclonal antibodies to different regions of the cystic fibrosis transmembrane conductance regulator (CFTR): detection of immunologically related proteins. J Cell Sci 108 (Pt 6):2433-44 (1995)): Wash cells 3x PBS, then fixe and permeabilize by incubation in methanol at -20°C for 5 min. Incubate coverslips in PBS/0.2% BSA for 5 min followed by a PBS wash and incubation with primary antibody in PBS/0.2% BSA for 1 h at room temperature. After washing in PBS/0.2 % BSA to remove excess primary antibody, incubate cells in secondary antibody. Please let me know if the customer still has problems with this protocol and I can offer you a replacement vial or credit note,

Thank you for contacting us for some advice on ab2916 and ab2784. You are correct, both antibodies were made using the same immunizing peptide, however they are raised in different species and more importantly we do not know if they recognise the same epitope within aa103-117, so a different epitope could explain the difference. We have not tested ab2916 in WB and from your feedback it does appear that it does not work in that application, would you please consider leaving a review on this antibody so we can add this information for future users? We reward reviews (negative and positive) with 100 Abcam points which can be used for a number of gifts. I hope the above information will help you, please do not hesitate to contact me again if you require further assistance,

Both ab2916 and ab2784 are made from the same synthetic peptide corresponding to amino acid residues 103-117. Ab2784 corresponds to amino acid residues 103-117 found in the first extracellular loop of human and rabbit CFTR. So, it appears that ab2916 was made from the extracellular portion. I hope this information helps; if you have any additional questions, please contact us again.

I'm sorry to hear you are experiencing problems with ab2916. This antibody has not been tested for western blotting, it is therefore difficult to find out what the problem might be. A litterature search about CFTR enabled me to find that the lysis buffer used to extract the protein from K562 cells is the following NP40 based buffer: 50 mM HEPES, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF, and 1 mM DTT. This information was from the reference "CFTR in K562 human leukemic cells. Assef YA, Damiano AE, Zotta E, Ibarra C, Kotsias BA. Am J Physiol Cell Physiol. 2003 Aug;285(2):C480-8." The article continues: "Nuclei and unbroken cells were removed with centrifugation (6,000 g for 15 min at 4°C). Total protein in each sample was quantified using the bicinchoninic acid assay (BCA Protein, Pierce Chemical). For Western blot studies, 100 ug of total proteins from K562 cells and 50 ug of total proteins from T84 cells were dissolved in loading buffer (4% sodium dodecil sulfate, 0.125 M Tris-HCl, pH 6.8, 0.2 M dithiothreitol, 0.02% bromophenol blue, and 20% glycerol). The preparation was heated to 90°C for 2 min, resolved on 7.5% polyacrylamide gel, and electrotransferred onto nitrocellulose membranes(Hybond ECL, Amersham Pharmacia Biotech, UK)." I would recommend trying a range of dilutions (e.g. 1/100-1/1000) and incubating the antibody overnight at 4C. I hope the above information will help you, please let me know if you require further assistance,