Troubleshooting ReadyTector®
Contents
1. Troubleshooting ReadyTector Appearance of smear on the The transfer buffer has not been washed out Optimize the washing step membrane completely before starting incubation with the Note that the membrane is fully moistened and free antibodies floating in the Wash Buffer If necessary please increase shaking during washing There are black spots on the Appearance of black spots on Western blotting Use the ReadyTector solution as described and with the membrane membranes is a common problem The reasons are__ corresponding ReadyTector Wash Buffer and ReadyTector multifactorial and can only be solved by optimizing Chemiluminescent Substrate all working steps Choice of substrate is important Use enough solution for the washing and incubation steps to enable the membrane to float in the solution In this way spots can be mostly reduced The ReadyTector Chemiluminescent Substrate has been optimized to reduce the number of spots in comparison to most other substrates But separate spots cannot be excluded Appearance of background on Washing has not worked Always wash carefully with the special ReadyTector Wash the membrane Buffer Note that the membrane is fully moistened and free floating in the Wash Buffer Shake the membrane during washing so that the membrane can freely float in the solution Wash 4 times after incubation with the antibody containing ReadyTector solution Blocking has not worked due to incorrect washing Opt2. cules of the canine serum This leads to additional bands READYTECTOR
3. ibody against a better antibody with higher affinity If this is not possible extend the incubation time up to 2 hours or more If the result is still bad switch to the standard procedure and forget the fast one step immunodetection with ReadyTector Some antibodies are not useful for fast immunodetection just due to low affinity of their binding Check your protein solution the gel run and the blotting procedure Check if your protein has been transferred to the membrane If possible change the antibody to solve the problem Ano ther possibility is to switch to the standard immuno detection protocol and to use LowCross Buffer as an antibody dilution buffer LowCross Buffer minimizes low to medium affinity binding and reduces non specific bands The secondary antibody binds proteins of the For detection of proteins in canine serum please use the specimen standard protocol with a secondary antibody pre adsorbed The specimen consists of canine serum The inclu against canine With ReadyTector Anti Mouse HRP the ded secondary antibody in ReadyTector recognizes additional bands cannot be prevented specifically the wanted species e g anti mouse identifies mouse But sometimes cross reactivities with rarely used species can occur This happens with anti mouse and canine serum If you separate and blot serum from dogs the secondary antibody included in the ReadyTector Anti Mouse HRP solu tion detects IgG mole
4. imize washing step See above step before incubation with the antibodies Blocking has not worked Use an adequate volume of ReadyTector solution for in cubation of the membrane Ensure that the membrane is fully moistened and free floating in the solution 20 mL for a membrane with the dimensions 8 x 10 cm is sufficient Less solution can cause problems READYTECTOR The membrane has been too long exposed to X ray film or imager after detection with the substrate No visible bands The membrane The primary antibody binds only with low affinity to doesn t show background the target Thus it is not able to bind the target protein in ReadyTector The blocking has worked and so no background is visible on the membrane The protein is not on the membrane The desired bands are ok but The antibody binds not only the target protein but there are additional bands also other proteins visible ReadyTector reduces non specific binding of low affinity Therefore the additional bands are caused by real binding of the antibody The primary antibody identifies specifically other proteins READYTECTOR Reduce exposition time In most cases the background will be lower with ReadyTector compared to the standard protocol The ReadyTector Chemiluminescent Substrate is a sensitive and fast substrate Thus exposition times can be reduced Instrument settings have to be adjusted The best solution would be to change the primary ant
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