Run a control, omitting the primary antibody incubation. If this information is not supplied by the manufacturer, optimize incubation time through testing.Įnsure the primary antibody is specific only for the protein that is being targeted. Test a range of concentrations to find optimal condition for individual assay.Ĭheck manufacturer's protocol to ensure the correct incubation time and temperature is used. If so, purchaseįor ECL method, ECL developing solutions are old/expiredįor ECL method, film exposure time is too shortįor ECL method, cling film interfering with reactionĬheck manufacturer's recommendations for primary and/or secondary antibody concentrations. If not, purchaseĮnsure film is not expired or exposed. Make sure the developing reagent being used is compatible with the enzyme conjugate.Įnsure substrate is made correctly by following the manufacturer's instructions.ĭecrease amount of salt present in wash buffer.ĭecrease amount of salt present in antibody dilution buffer.Įnsure the developing reagent was prepared correctly.Ĭheck that the reagent is still active. Use a washing buffer that does not contain a detergent, or that has a lower concentration of detergent present. Shorten wash times and/or reduce number of washes. Increase incubation period of secondary antibody (please see manufacturer's recommendations for incubation time and temperature). Insufficient incubation of secondary antibody Antibody can be incubated for at least one hour at room temperature or overnight at 4☌. Increase incubation period of primary antibody. Insufficient incubation of primary antibody Rabbit-anti-humanĮnsure the secondary antibody being used is against the species of the primary antibody (i.e. Test a range of concentrations to find the optimal condition for individual assay.Įnsure primary antibody reacts with the target protein from the species being studied (i.e. Insufficient amount of primary and/or secondary antibodyĬheck manufacturer's recommendation for antibody concentration. Perform a Dot Blot to ensure antibody is still active. If antibody was subjected to repeated freeze/thaw cycles purchase new antibody, as this can affect the structure and protein:antibody binding. If expired or past storage time, purchase new antibody. Make sure buffers do not contain sodium azide, as this will interfere with the HRP signal.Ĭheck manufacturer's recommendations for storage and/or expiration date. We recommend 3% Non-Fat Dry Milk in diH2O. Optimize concentration of protein in blocking buffer. Substitute buffer with a higher pH buffer (i.e. The presence of SDS during transfer can reduce the binding of the protein when using a nitrocellulose membrane.Īdd 20% methanol to transfer buffer to increase binding.Ĭheck protein transfer by staining the membrane with a reversible stain, such as Ponceau S, to visualize major bands.
(Please see our Western Transfer protocol for correct transfer "sandwich" set up.)Įnsure the correct transfer buffer is being used. Make sure transfer "sandwich" is set up correctly and there is sufficient contact between gel and membrane. Make sure membrane is pre-soaked according to manufacturer's instructions.Ĭheck and optimize transfer time, as appropriate transfer time will vary with MW of the target protein. Protein over-transferred/did not transfer correctlyĭecrease voltage for lower MW proteins (less than 10kDa). Confirm protein is present through an alternate method.ĭecrease running time and monitor the gel by watching dye front and ensuring the current is turned off when dye front is near bottom of gel.
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