Quantitative immuno-slot blot assay to measure ACMPK protein in cell extracts of Aspergillus nidulans.

A primary means of detecting and identifying specific proteins in cellular extracts is by Western blot analysis (4,13). Quantitation of a protein of interest is generally accomplished using an enzyme-linked immunosorbent assay (ELISA; References 8, 9 and 11) or radioimmunoassay (4,8). When ELISA or radioimmunoassay procedures for the protein of interest are unavailable, densitometric analysis of Western blots is often used to estimate the amount of antigen present (3). However, quantitation of proteins on Western blots is subject to factors that limit the ability to accurately estimate the amount of the specific protein in the samples. These include variations in protein loading and loss of protein during electrophoretic transfer. Often, quantitation is performed on a single aliquot of the sample, with no indication of reproducibility of the result (3). Slotand dot-blotting techniques serve to overcome some of these limitations because multiple aliquots of the samples are directly applied to the membrane, eliminating the need for the transfer of protein (6–8). Slot blotting is more conducive to accurate densitometric measurements than dot blotting and is generally preferable for work of a quantitative nature. However, quantitation by densitometric scanning has inherent limitations if care is not taken to ensure that the signals are within the linear range of response of the method of detection. This includes scanning of autoradiograms obtained using radioactive or luminescence-based detection reagents where all of the samples might not lie within the linear range of response of the photographic film. We have developed a slot-blot immunoassay to quantitate the amount of Aspergillus nidulans calcium/calmodulindependent multifunctional protein kinase (ACMPK) present in cell extracts of this filamentous fungus (1). We describe the development of the assay and its use to detect the amount of ACMPK protein present in extracts prepared from cell cycle phase-specific and developmental phase-specific cultures of A. nidulans. Previous attempts to develop and perform ELISAs or quantify this protein by Western blot analysis have met with only limited success, as indicated by a high degree of variability from assay to assay. The use of the immuno-slot blot assay has yielded reproducible results, allowing for the quantitation of this protein in different cellular extracts. Homogeneous cell cycle phase-specific cultures were prepared using A. nidulans strain SO6 (nimA5; wA2; yA2; chaA1; pyrG89; cnxE16; choA1). A. nidulans strain SO6 carries the temperature-sensitive nimA5 mutation. Cells carrying the nimA5 mutation become blocked in late G2 phase at the restrictive temperature (42°C). Upon return to permissive temperature (32°C), cells enter mitosis within 5 min (2). Homogeneous G1-, S-, G2and Mphase-specific SO6 cultures were prepared from cells that were initially grown for 16 h at the permissive temperature (32°C). S-phase cultures were prepared by shifting the cells to 42°C for 4 h and then transferring to medium containing 20 mM hydroxyurea for 90 min at 32°C. The G2-phase cells were shifted to 42°C for 4 h. M-phase cells were prepared by shifting cultures to 42°C for 4 h, followed by a 30-min incubation in 5 μg/mL benomyl at 32°C to block cells in mitosis. The G1-phase culture was prepared by shifting cells to 42°C for 4 h and then transferring to 32°C for 15 min. We were also interested in measuring ACMPK protein levels during the process of conidial germination and mycelial growth in A. nidulans. R153 (wA2; pyroA4) conidia were inoculated into 500 mL defined medium containing 0.2% Tween 20 to a final concentration of 2.0 × 107/mL. The conidia were allowed to germinate, and the culture was incubated for 16 h at 37°C with shaking. Samples were collected at set time points (2, 3, 4, 6, 8, 10, 12, 14 and 16 h). During this time period, A. nidulans cells developed from uninucleate conidia to long-branched, multinucleate mycelia (12). Cell extracts were next prepared as follows: (i) frozen cells were ground in a mortar with a pestle in liquid nitrogen and 2 mL/g tissue of 25 mM Tris-HCl, pH 8.0, 15 mM EDTA, 0.2% Nonidet P-40 (NP40), 1 mM diisopropylfluorophosphate, 10 μg/mL leupeptin; (ii) homogenates were centrifuged at 14 000× g for 15 min at 4°C; (iii) the pellets were discarded; and (iv) the extracts were stored at -90°C. Protein content of all samples was determined by the method of Schaffner and Weissmann (10). Cell extracts were assayed for ACMPK protein using the immunoslot blot protocol developed. Aliquots of cell extracts were diluted with 100 μg/mL bovine serum albumin (BSA) to a final volume of 300 μL. For each ex-