Bio Rad Slot Blot Apparatus
Western blotting is a widely used technique by research and clinical labs to detect proteins with their specific antibody. Blots require an apparatus, or blotter to transfer the gel to membrane under wet, dry or semi-dry conditions. Transfer protocol depends on type of protein, gel thickness, and type of membrane. Electroblotting systems feature easy blotting of multiple gels simultaneously in less than an hour. Mini and large tank systems produce uniform and consistent results. Semi-dry blotting systems offer fast and efficient transfers of proteins to membranes with low buffer volume. Most blotting apparatus include essential accessories. Slot-blot assay The slot-blot method was used to determine levels of protein 3-nitrotyrosine (3-NT) in brain as previously described 37,38. For 3-NT determination, samples were solubilized in Laemmli buffer. Protein (250 ng) from each sample was loaded onto a nitrocellu-lose membrane in respective wells in a slot-blot apparatus (Bio-Rad.
Overview
Test blots, as their name implies, are very simple Western blots that are created for the express purpose of optimizing or troubleshooting experimental conditions. They are usually produced by running multiple lanes of the same lysate or purified protein solution on a gel, and after transfer cutting the blot into strips to be tested individually.
They provide a quick and efficient means of examining a range of antibody dilutions or detection substrates
Slot-blot analysis was performed to compare Epo-R content. All samples analyzed were previously shown to be without evidence of degradation. Five ug RNA were denatured with a solution of 10 m.MNaOH and 1 imi EDTA, and loaded onto nylon membrane (Zeta-probe) placed in a slot-blot apparatus as described in the instruction manual (Bio-Rad). Bio-Rad's fluorescent western blotting workflow is a seamless integration of products designed to work together to offer guaranteed results. This validated set of solutions will make it easy for you to get better data every time.
Dot blots and slot blots are also a very useful variation on the typical Western blot. They do not require gel electrophoresis, so there is no separation of proteins by size.
Instead, the target protein or cell lysate mixture is added directly onto the surface of the nitrocellulose or PVDF membrane. Protein solutions can be applied directly in a small volume, or with a vacuum manifold to produce an orderly grid of samples similar to that seen in Figure 14. Each dot or slot blot would contain known amounts of target protein or cell lysate.
Once dry, dot blots and slot blots are subjected to the same immunodetection steps used for Western blotting, i.e. blocking, antibody incubation, and target detection with substrate. Grey and black spots on the figure below indicate which samples are positive for the target protein and correspond roughly to the bands produced on a Western blot.
Figure 14: Dot Blots and Slot Blots. The dot blots at the left represent a dilution series of a sample, with smaller lighter dots corresponding to lower concentrations of target protein. The slot blots represent a group of random samples, the intensity of the signal corresponds to the concentration of the target protein in that sample.
Dot Blot
By making a number of identical dots or slots of a single known protein sample, or a range of sample dilutions, one can quickly test several combinations and concentrations of primary and secondary antibodies. Dot blots and slot blots are also beneficial when screening a large number of samples, or if a simple answer will suffice.
Bio Rad Slot Blot Apparatus Diagram
The main downside to slot blots/dot blots is that they provide no information about molecular weight. Thus, it is harder to detect false positive signals, or to tell whether modified forms of a protein are present.
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