2011-2012 Catalogue - PeproTech, Inc.

www.peprotech.com2011-2012 CatalogueWestern Transfer Troubleshooting Guide ContinuedINFORMATION & TECHNICAL SUPPORTProblem Possible source SuggestionSplotchy background Uneven agitation during incubations • Ensure the membrane is agitated evenly byˇplacing on a rocker or shaker during incubation.ˇ Insufficient amount of incubation • Ensure enough solution is present during eachˇ buffer incubation period to fully submerge theˇmembrane and allow it to float freely in theˇsolution.ˇ Air bubbles present during transfer • Gently remove all air bubbles from transferˇ“sandwich” before transfer is started. This can beˇdone by using a roller or clean glass rod/Pasteurˇpipette.ˇ Membrane problems • Ensure membrane is wetted thoroughly accordingˇto the manufacturer’s protocol.ˇ• Handle membranes with extreme care.ˇMishandled membranes can cause non-specificˇbinding.ˇ• Handling membranes with bare hands can lead toˇcontamination of membrane. Use forceps or wearˇgloves when handling membrane.ˇ Aggregation of HRP conjugate • If using HRP, filter conjugate to remove HRPˇaggregates.ˇ• Use a fresh sample of HRP conjugate.ˇ Contamination of reagents • Filter buffers before use to remove contaminant.ˇ• Make fresh buffers and re-run Western Transfer.Non-specific bands Antibody concentration too high • Check manufacturer’s recommendations forˇprimary and/or secondary antibody concentrations.ˇ• Test a range of concentrations to find optimalˇcondition for individual assay.ˇ Old or degraded antibody • Check manufacturer’s recommendations forˇstorage and/or expiration date. If expired or pastˇstorage time, purchase new antibody.ˇ• If antibody was subjected to repeated freeze/thawˇcycles purchase new antibody, as this can affectˇthe structure and protein:antibody binding.ˇ Non-specific binding of primary • Decrease concentration of primary antibody.ˇ antibody • Check concentration of protein being added to gel.ˇ• Use a more specific antibody, such as aˇmonoclonal antibody or Antigen Affinity Purifiedˇpolyclonal antibody.ˇ• Add a small amount of detergent, such asˇTWEEN-20 (0.1-0.5%) to the primary antibodyˇsolution and/or blotting buffer.ˇ• Increase number/duration of washes.ˇ Non-specific binding of secondary • Decrease concentration of secondary antibody.ˇ antibody • Run a control, omitting the primary antibodyˇincubation. If bands develop, choose a differentˇsecondary antibody.ˇ• Add a small amount of detergent, such asˇTWEEN-20 (0.1-0.5%) to the secondary antibodyˇsolution.ˇ• Increase number/duration of washes.170To Order: EC: Tel: +44 (0)20 7610 3062 • email: info@peprotech.co.ukBestellungen: AUSTRIA: Tel: +43 (0)1 405 9696 • email: info@peprotech.atObjednávky: Česká Rep.: Tel: +420 225 992 284 • email: info@peprotech.cz