The procedure illustrated above is a general initial step, described in the majority of Neurospora protocols, necessary for the production of the starting material from which proteins, RNA, DNA, and so on can be extracted. This section describes the protein extraction and immunoprecipitation steps. Here the general techniques of cell lysis and protein extraction and purification are carried out using buffers that specifically preserve the PKC catalytic activity.
1. Weigh 200 mg of mycelial powder in a vial and resuspend in 1 mL of ice-cold protein lysis buffer.
2. Gently blend the mycelia in lysis buffer with a homogenizer. This step is necessary to destroy the cell walls and release the proteins.
3. Pellet the debris by centrifugation at 12,000g for 15 min at 4°C, move the supernatant (containing the proteins) to a clean vial (see Note 9).
4. Immunoprecipitate PKC incubating the protein lysate with 50 pL of FLAG M2 antibody resin resuspended 1:1 in lysis buffer (50% slurry). The incubation is carried out for 3 h at 4°C on a rotatory wheel at the lowest speed.
5. Precipitate the beads by centrifugation at 10,000g for 10 s. Replace the supernatant with 1 mL of lysis buffer. Repeat this wash two more times.
6. Precipitate the beads by centrifugation at 10,000g for 10 s. Resuspend the beads in 200 pL of lysis buffer containing 100 pg/mL of FLAG peptide to elute PKC (see Note 10).
7. Precipitate the beads by centrifugation at 10,000g for 20 s and move the supernatant containing the eluted kinase to a clean vial. The eluted PKC can now be used to perform the phosphorylation reaction.
Fig. 1. Immunopurified protein kinase C (PKC) phosphorylates myelin basic protein (MBP). Top panel: phosphorylated PKC. Middle panel: phosphorylated MBP. Lower panel: total PKC protein levels obtained by immunoprecipitation.
1. To measure the activity of the eluted kinase mix: 40 ^L of eluted kinase, 12 ^L of 5X PKC reaction buffer, 1 ^L of 1 mg/mL MBP, 5 ^Ci [y-32P]ATP, H2O to 60 ^L. Incubate at 30°C for 30 min.
2. Stop the reaction by adding 60 ^L of 2X Laemmli buffer.
3. Denature the proteins by incubating at 95°C for 5 min and separate by running on a 7.5% SDS-PAGE. Usually the whole reaction is loaded on the gel. For optimal separation of the protein bands, the use of a large protein gel apparatus (i.e., Hoefer) is recommended.
4. Wrap the gel in ceramic wrap and analyze in a phosphoimager. The bands detected correspond to proteins (present in the phosphorylation mix) that have incorporated radioactive ATP. This reaction was mediated by the activity of the immunopurified kinase; thus, keeping constant every other condition, the intensity of the bands is proportional to the activity of the kinase. This allows assessment of the activity of the kinase produced under different physiological conditions. In the case where the kinase is active, two bands should appear—the MBP and the autophosphorylated PKC—whereas in the case where the kinase is not active, no bands or lower intensity bands of both MBP and PKC are expected
1. See the website of the Fungal Genetics Stock Center, www.fgsc.net/fgn41/ campbell.html, for a description of the pMYX2 plasmid.
2. To prepare 1 L of Vogel's 50X salts, dissolve in 750 mL of distilled water (at room temperature) the following (in this order): 125 g Na3 citrate-2H2O, 250 g KH2PO4 anhydrous, 100 g NH4NO3 anhydrous, 10 g MgSO4-7H2O, 5 g CaCl2-2H2O, 5 mL of trace element solution (see below), 2.5 mL of biotin solution. Add 2 mL of chloroform as a preservative and store at room temperature. To prepare 100 mL of trace element solution, dissolve in 95 mL of distilled water (at room temperature) the following (in this order): 5 g citric acid-1H2O, 5 g ZnSO4-7H2O, 1 g Fe(NH4)2(SO4)2-6H2O, 0.25 g CuSO45H2O, 0.05 g MnSO41H2O, 0.05 g H3BO3 anhydrous, 0.05 g Na2MoO4-2H2O. Add 1 mL of chloroform as a preservative and store at room temperature. The biotin solution is prepared by dissolving at room temperature, 5 mg of biotin in 50 mL of distilled water. Aliquot and store at-20°C.
3. Add the protease inhibitors phenylmethylsulfonyl fluoride, leupeptin, and pepstatin immediately before use.
4. Neurospora growth conditions may vary slightly from one laboratory to another, depending on the requirements of each experiment. Among the variables are the storage conditions of the conidia. Most experiments require that exactly the same growth conditions are used for all tests done, and it is well known that cycles of freezing and unfreezing of the conidia stored in water reduce the viability of the conidia dramatically. This results in variability of the number of viable conidia inoculated for each experiment. It is, therefore, recommended to prepare aliquots of the stock and test the viability of the conidia for each experiment. It is alternatively possible to use freshly harvested conidia for each experiment, isolated from Neurospora growing in solid media.
5. The selection is given by the presence of 100 ^g/mL benomyl, a fungicide to which the strains transformed with the pMYX2fK plasmid are resistant.
6. To induce the qa2 promoter on the pMYX2fK plasmid and the expression of PKC, the growing mycelia are incubated in the presence of 0.03% quinic acid. The activating effect of the quinic acid on the qa2 promoter is inhibited by the presence of the carbohydrate contained in the minimal medium. To prevent this inhibitory effect, the mycelia are filtered and then resuspended in minimal medium without sucrose, containing 0.03% quinic acid.
7. Expression levels induced by the qa2 promoter are very variable and can be very different in transformants generated from the same plasmid. This is why transformants are usually screened for the preferred type of expression: highest, stringent, low, and so on. When a vector is transformed into Neurospora, it is randomly integrated into the genome by nonhomologous recombination. Thus, insertions occur by chance in heterochromatic or euchromatic regions, which explains the consequent variability of expression. It is therefore recommended to test the expression level of recombinants every time, preferably by Western blot ting, as it is necessary to have high amounts of kinase to perform the kinase assay.
8. It is very important to grind the mycelia very finely, as more proteins will be extracted. However, once the mycelia are grinded they can more easily thaw and it is important to always keep the vials in liquid nitrogen. All subsequent steps are on ice or at 4°C; this is to preserve the activity of the kinase as close as possible to its original state. This is particularly important when the kinase activity is tested in specific conditions (such as light, dark, different circadian times, etc.). Also, mycelia are sometimes stored for longer than 1 mo at -80°C, especially when not needed for delicate experiments; however, it is recommended not to store the samples for too long.
9. This protocol is based mostly on mechanical disruption of the cell, so that it becomes very important to accurately grind and homogenize the mycelia. However this does not favor the isolation of membrane associated proteins. It is known that PKCs are usually found associated to the plasma membrane; therefore, here we add the detergent Triton-100X to the lysis buffer to a final concentration of 0.05%. Higher concentrations would favor the isolation of a higher amount of membrane protein, but it would decrease the efficiency of the following immu-noprecipitation. It is therefore important to empirically find the Triton concentration that allows for purification of sufficient amount of PKC and that does not interfere with the immunoprecipitation.
10. Higher amounts of eluted kinase would be obtained by resuspending the pellet in larger volumes, and repeating the step two or more times. This would result in very large final volumes of low-concentration purified kinase. As it is not recommended to perform the phosphorylation assay in very large volumes, it is important to obtain the purified kinase in a highly concentrated small volume. We find that the elution step described here results in an amount of purified kinase sufficient to perform at least five phosphorylation assays. It is important to verify the success of the immunoprecipitation by Western blot.
1. Liu, Y. (2003) Molecular mechanisms of entrainment in the Neurospora circadian clock. J. Biol. Rhythms 180, 195-205.
2. Vogel, H. J. (1956) A convenient growth medium for Neurospora. Microb. Genet. Bull. 13, 42-43.
In Vitro Systems
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