Specific Detection of Native Multi-Subunit Proteins by Slot-Blot Assay

Detection of membrane-bound proteins by immunoblot (Western blot) is by far the most commonly used procedure for detection or quantitation of an antigen (11). However, since this technique requires analysis of the antigen on a denaturing gel using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the antigen generally loses its native conformation (7). Although resolution can be performed on a non-denaturing gel, neutral or positively charged proteins from such gels do not transfer well electrophoretically. Moreover, all kinds of gel electrophoresis is timeand labor-intensive. We describe a simple and quick procedure for detection of proteins adsorbed directly on membranes using a slot-blot apparatus, followed by probing of the membrane with an appropriate reagent specific for the immobilized protein. We have routinely used Immobilon-P (Millipore, Bedford, MA, USA) with success, although related membranes such as Immobilon-Psq worked equally well (especially for smaller proteins; data not shown) and can be optimized for specific proteins. We have successfully detected a number of multi-subunit proteins that required interaction between the subunits such as enzymes and immunoglobulins. The enzymes could be detected by activity assays and the IgGs detected through the binding of antigens, suggesting that the majority of the membrane-bound proteins might retain their native conformation to a large extent. Thus, at least in principle, a variety of other probes could also be used for detection, including an antibody or a binding protein that is visualized by standard detection systems based on radioactivity, color or emission of light, some of which have been demonstrated in this paper. Furthermore, if needed, the system is easily amenable to automation. The procedure was developed while we were trying to purify an antibody against respiratory syncytial virus (RSV) phosphoprotein P from an immune-rabbit serum; although, as shown later, we have extended its use to a number of other multi-subunit proteins, including enzymes. The rabbit antiserum was subjected to the standard procedures of precipitation with ammonium sulfate followed by chromatography over DEAE cellulose (6). The bound antibody was eluted with a gradient of 50–500-mM NaCl solutions; the fractions were analyzed by SDSPAGE followed by staining, with the hope that we would be able to recognize the heavy (55 kDa) and light chains (25 kDa) of the IgG by their mobility with respect to standard protein markers. The stained gel pattern in Figure 1 shows that protein bands are discernible in lanes 4–28 and that the mobility of the major band roughly corresponds to the size range of the IgG heavy chain. Unfortunately, it also migrated too closely to the bovine serum albumin (BSA) marker, and we were not sure whether it represented rabbit albumin or IgG. The light chain did not stain well enough to be used as a criterion in selecting the fractions. Thus, we wanted to develop a quick, easy and specific procedure to monitor the presence of IgG in a large number of fractions in a short time. Although the IgG could be detected by standard immunoblot procedures using a secondary anti-rabbit antibody against either the heavy or the light chain, we also wanted to use 32P-labeled P protein (the antigen) as a probe for a more specific detection. This led us to the development of the method as detailed below. Immobilon-P membrane of appropriate size was first soaked in absolute methanol for 2 min as advised by the manufacturer and then washed with 50 mL of Buffer A (50 mM Tris-HCl, pH 8.0, 50 mM NaCl) for about 1 min. The washing was repeated 4× to remove the methanol completely. A standard slotblot apparatus (Hybri-Slot Manifold; Bethesda Research Laboratories, Bethesda, MD, USA) was assembled with this membrane. Various amounts (1–5 μL) of the relevant column fractions were diluted to 200 μL in Buffer A, and the diluted proteins were loaded in the slots. Proteins were adsorbed to the membrane by slow suction of the liquid using a water aspirator pump (about 1–2 min). Each well was immediately washed twice with 400 μL of Buffer A.