2011-2012 Catalogue - PeproTech, Inc.

www.peprotech.com2011-2012 CatalogueWestern Transfer FAQINFORMATION & TECHNICAL SUPPORT1. How long will the immunostaining take whenPeproTech’s Western Transfer protocol isfollowed?The Western Transfer process will takeapproximately 6 hours from transfer of proteinsto visualization of bands.2. What type of molecular weight markershould be used?PeproTech uses Invitrogen’s Multi-Mark prestainedmolecular weight marker for use in allWestern Transfers performed in-house, althoughthis molecular weight marker does not have tobe utilized. However, it is necessary that a prestainedmolecular weight marker is used whennot utilizing the ECL detection method.3. Is the addition of a positive control necessary ina Western Transfer?Yes, in order to know exactly how the proteinof interest will visualize on the Western Transferyou must add a positive control to your gel.When using PeproTech’s antibodies in a WesternTransfer, we recommend using the correspondingPeproTech recombinant protein, which was theimmunizing antigen for the antibody of choice.4. Does the color development system recommendedin PeproTech’s Western Transferprotocol have to be used?No. There are many different color developmentsystems that can be used for the visualizationof your Western Transfer. However, thesystem that is chosen must be compatible withthe enzyme conjugate being used. PeproTechuses an alkaline phosphatase linked secondaryantibody. NBT/BCIP is suitable for use with thisenzyme, and is therefore used for the visualizationof our Western Transfers.5. Is agitation of the membrane essential during theincubation periods?Yes, it is essential that the membrane beagitated during the incubation periods. If themembrane is not agitated the antibodies,blocking buffer, and washing buffer may notaffect the membrane evenly and can createsplotchy or patchy background. It can also limitthe detection of the proteins by the antibodies.6. Why are some Western Transfer resultsstronger than others when using different lots ofthe same antibody?Due to the nature of polyclonal antibodies,variability may be seen from lot to lot.7. Is it necessary to include the filter paperduring the protein transfer step?Yes, it is necessary to include the filter paperas a barrier in the protein transfer step. This filterpaper helps protect the gel and membrane fromany possible damage resulting from directcontact with the sponges, yet does not interferewith the electric current.8. Can an enzyme-conjugated primary antibody beused in a Western Transfer rather than using aprimary/secondary antibody system?Yes, an enzyme-conjugated primary antibodycan be used instead of utilizing a primary/secondary antibody system in your WesternTransfer. However, by using a labeled secondaryantibody that recognizes the antigen specificprimary antibody, there will be an amplificationof the signal seen in a Western Transfer whencompared to using the enzyme-conjugatedprimary antibody alone.166To 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