2011-2012 Catalogue - PeproTech, Inc.

PEPROTECH ®www.peprotech.comProblem Possible source SuggestionNon-Specific Bands Aggregation of target protein • Add or increase a reducing agent, such as DTT orcontinuedBME, to ensure all disulfide bonds are reduced.ˇ • Heat protein sample in a hot water bath for 5-10ˇminutes before loading onto gel.ˇ• Check concentration of protein being added toˇgel. A concentration that is too high can causeˇprotein aggregation. Re-run Western Transferˇwith a lower concentration of protein.ˇ Degradation of protein sample • Make fresh samples and minimize freeze/thawˇcycles of samples.ˇ• Add protease inhibitor to samples prior to storage.ˇ• Keep samples stored between -20°C and -80°C.ˇ Insufficient blocking • Test a different blocking buffer formulation.ˇ• Batch of blocking buffer may be inactive. Use aˇfresh blocking buffer.ˇ• If blocking overnight at 4°C, this may decreaseˇblocking efficiency since lower temperatures mayˇlessen the effectiveness. Try incubation for oneˇhour at room temperature.ˇ• Add TWEEN-20 to blocking buffer.ˇ• Optimize incubation time and/or temperature ofˇblocking step.ˇ SDS facilitates binding to • Ensure membrane is sufficiently washed afterˇ immobilized protein bands transfer of proteins.ˇ• Do not use SDS during immunostaining whenˇusing a nitrocellulose membrane.ˇ Excess enzyme present • Reduce concentration of the selected enzymeˇconjugate.ˇ Contamination of reagents • Filter buffers before use to remove contaminant.ˇ• Make fresh buffers and re-run Western Transfer.Diffused/Fuzzy 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 Excess protein on gel • Further dilute protein samples before loading onto gel.ˇ Gel over-heated/high voltage • Heat causes the gel to lose its structure andˇresolving power. Be sure the transfer reservoir isˇrun in an ice bath.ˇ• Reduce voltage or current used during transfer.ˇ Incorrect preparation of membrane • Pre-soak membrane in correct transfer solutionˇ(see membrane manufacturer’s protocol). Ensureˇit is soaked for the correct amount of time.White Bands Antibody or protein concentration • White bands are the result of rapid consumptionˇ(ECL visualization too high/Excessive signal generated of the substrate caused by the presence of tooˇmethod)much conjugate. This is the result of highˇconcentrations of the antibodies and/or proteins.ˇCheck manufacturer’s recommendations forˇantibody and/or protein concentrations.INFORMATION & TECHNICAL SUPPORTBestellungen: Deutschland: Tel: 0800 436 99 10 • email: info@peprotech.dePour commander: France: Tel: +33 (0)1 46 24 58 20 • email: info@peprotechfr.comTo Order: NORDIC: Tel: +46 (0)8 640 41 07 • email: info@peprotech.se171