Two-fold serial dilutions of HeLa cell lysate (20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 µg) were separated by SDS-PAGE and transferred to nitrocellulose (panels A–C) or PVDF (panels D–E) membranes. For example, in applications where alkaline phosphatase conjugates are used, a blocking buffer in TBS should be selected because PBS interferes with alkaline phosphatase.Ĭomparison of SuperBlock Blocking Buffer and milk. The proper choice of blocker for a given blot depends on the antigen itself and on the type of detection label used. The blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal-to-noise ratio. A variety of blocking buffers ranging from milk or normal serum to highly purified proteins have been used to block free sites on a membrane. Renaturation of the protein also occurs during the blocking step, so it is important to optimize the blocking conditions to obtain the best signal-to-noise ratio for each application. Insufficient blocking may result in high background, whereas prolonged blocking could result in a weak or masked signal. Bovine serum albumin (BSA) is often used as a starting point for many membrane-probing reactions. The determination of an effective blocking buffer must be made empirically. Any given protein blocker may crossreact or otherwise disrupt the target protein–protein interaction. A variety of different protein blockers may be used, and no one blocking protein solution will work for all blotting experiments. By blocking all remaining binding sites on the membrane, a blocking buffer reduces nonspecific binding and aids in protein renaturation. Other labeling and detection schemes are possible.Īfter transferring proteins to the membrane, western blotting procedures require that unreacted binding sites on the membrane be blocked with a non-relevant protein solution. By contrast, bait proteins generally are not enzyme labeled because a large enzyme label is likely to sterically hinder unknown binding sites between bait and prey proteins. †Labeled antibodies are generally labeled with an enzyme (either horseradish peroxidase or alkaline phosphatase). This general approach can be adjusted, as shown in the table below, by using untagged bait protein that is detected by antibody, biotinylated bait protein that is detected by enzyme-conjugated streptavidin, or radiolabeled bait protein that is detected by exposure to film. Once bound, enzyme (horseradish peroxidase HRP)-conjugated antibody that targets the bait tag is used to label the interaction, which is then detected by enzymatic chemiluminescence. In this example, a tagged bait protein is used to probe either the transfer membrane or a gel for the prey protein. Following the reaction of the bait with the prey protein, a detection system, dependent upon the bait protein used, identifies the band that corresponds to the prey protein.ĭiagram of far–western blot to analyze protein–protein interactions. After transfer, the membrane is blocked and then probed with a known bait protein, which usually is applied in pure form alternatively, this reaction can be performed in-gel, as described later in this review. The sample (usually a lysate) containing the unknown prey protein is separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) or native PAGE and then transferred to a membrane. The far–western blot technique is similar to western blotting in a western blot, an antibody is used to detect the corresponding antigen on a membrane, while in a classical far-western analysis, a labeled or antibody-detectable "bait" protein is used to probe and detect a target "prey" protein on the membrane.
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