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Hereby I would like to thank you kindly for your help over the telephone. By providing you with some of my data I hope that you can help me answer the following question: How can I distinguish which of the two bands on my blot represents the 45 kDa band that matches the expected GLUT-4 band? Important notifications regarding the protocol that I use are stated here: - None of the samples where heated prior to gel electrophoresis - Antibody used: ab654 “anti-glucose transporter GLUT-4 antibody” (Abcam) (1/1000 dilution) [WB : Detects a band of approximately 45 kDa, predicted molecular weight: 54.8 kDa] - Over-night incubation with antibody - Sec antibody: anti-rabbit 1:1000 - WB Detection Kit: Amersham ECL™ Advance Attached to this e-mail you will find an image of my blot showing the problem that I experience. I appreciate the help and service that Abcam provides. Thank you in advance. Best Regards,
Asked on Nov 23 2011
Thank you very much for sending the image and for providing details about the experiment. The possible reason for appearance of doublet could be the different glycosylated or phosphorylated forms of GLUT4 or the lower band could itself be degradation form, The doublet 45 to 50kDa is shown in many publications so it will be worth checking the literature also http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1218126/pdf/9032457.pdf http://www.pnas.org/content/88/6/2525.full.pdf Three possible variation of the experiment can be tried 1. Trying blocking the antibody with a blocking peptide e.g. ab115831 and then checking the results 2. Trying a full length GLUT4 protein as positive control (we unfortunately do not have this product) 3.Trying a cell line lysates as positive control which express GLUT4 (Heart muscle lysates, ab29423, ab29431, skeletal muscle cell lysates ab29330, ab29320) I am presuming the protocol used is absolutely fine however I am taking this opportunity to suggest few updates that may make difference. Please ignore if you are already using these. - Use fresh protease inhibitors in lysis buffer. - Heat sample with loading buffer for at 60-70C for 10-15 minutes; do not boil the sample. - Try using 5% BSA for blocking which we normally use in our own lab and which for unknown reasons works better. I am sure these suggestions will help. Let me know if you want any help in ordering any of these suggested products. Please also do not hesitate to contact me if you have any additional questions.
Answered on Nov 23 2011