![]() ![]() Washes and antibody incubations can also be performed using the units. Include spaces containing no primary or secondary antibodies to control for backgroundĭot blot procedure using a commercial apparatusĬommercial dot blot apparatuses immobilize, concentrate and bind samples to membranes using a vacuum to draw the sample onto the membrane.Choose ranges of antibody dilutions that encompass the recommended concentration and two dilutions above and below the recommended concentration.Plan to titrate primary and secondary antibodies on opposing axes.Create a diagram of experimental conditions (similar to Figure 1).The following guidelines should be followed when planning the experiment: Figure 1 is an example of an experiment to titrate a primary antibody with a recommended dilution of 1:1000 and a secondary antibody with a recommended dilution of 1:10,000. Titration of both primary and secondary antibodies can be performed simultaneously by using a checkerboard titration pattern. If a sample contains precipitates, centrifuge the sample and only apply the supernatant to membrane to prevent clogging.If detergents are present, dilute the samples with buffer.Do not prepare samples in buffers containing detergents as they will inhibit binding of the protein to the membrane.Prepare enough sample in sufficient volume to accommodate all the conditions being tested.These apply both to when using microfiltration units or when spotting the protein manually. Negative control samples will determine whether any observed signal is due to non-specific cross-reactivity.Īlthough sample preparation for dot blotting is similar to sample preparation for traditional Western blotting, several factors should be kept in mind. Negative control samples can also be included, particularly if cell lysates are used. Recombinant protein is ideal, however cell lysates containing highly expressed protein can also be used. Protein samples for titrating antibodies should contain the protein of interest in abundance. The region containing each dot must then be individually excised and treated separately for incubations and washes. In this case, protein is spotted manually onto the membrane in a series of small dots. Microfiltration units provide an ease of use as protein blotting, incubations and washes can all be performed within the unit which isolates each individual blot.ĭot blots can also be performed without the aid of a microfiltration unit. However when the integrity and identity of the protein is known, the dot blot format can be used to provide substrates for titration of antibodies.Ĭommercially available units for dot blottingĭot blots can be performed using commercially available apparatuses, often called microfiltration units. Because proteins are not first separated using a gel, dot blots cannot be used to determine the molecular weight of a protein nor can they discriminate between alternate forms of the protein (e.g. Optimal antibody concentrations can be efficiently determined by adhering proteins to nitrocellulose using a dot blot technique with a checkerboard pattern to determine the optimal primary: secondary concentration pair.īelow are guidelines and general protocols for performing dot blots either by using a microfiltration unit or by manually spotting protein onto a membrane.ĭot blots are similar to Western blots, however the proteins are not separated electrophoretically prior to transfer to a membrane but are instead spotted directly onto a membrane. Although dot blots cannot determine the molecular weight or integrity of a protein and therefore should never be used to identify a protein per se, they are particularly useful in titrating antibodies. The best results for Western blots are obtained when both the primary and secondary antibodies are accurately titrated. ![]()
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