Using plant protein microarrays to study protein phosphorylation experiments
Identification of protein kinase substrates is a major focus of protein kinase research and provides evidence for resolving signal transduction pathways and their complex regulation. We present here an ex vivo experimental method based on protein microarray technology for systematic phosphorylation screening of proteins immobilized on microarrays with specific protein kinases. This high-throughput method can be used to identify possible kinase substrates. The source of this experiment is the "Guide to Plant Proteomics Experiments" [French] H. Tillemment, M. Zivi, C. Damerweil, and V. Michen, eds.
Operation method
Using plant protein microarrays to study protein phosphorylation
Materials and Instruments
Isopropyl β-D-thiogalactoside Move 3.1 Purification of recombinant kinases under nondenaturing conditions For more product details, please visit Aladdin Scientific website.
Non-denaturing lysate Non-denaturing wash solution Non-denaturing eluent Phenylmethylsulfonyl fluoride
Ultrasonic homogenizer Polypropylene column
The kinase used for the phosphorylation screen must be soluble and active and purified without the involvement of other kinases. Therefore, we can produce and purify histidine-tagged recombinant protein kinases in large quantities from cDNA expression libraries, including, for example, barley expression libraries constructed with the E. coli vector PQE30NST ( Retrieval No. AF074376;22 ) or with the pQE30NASTattB vector (Retrieval No. AY386205;23 ; See also 28.3.1 ) constructed Arabidopsis expression libraries. We expressed and purified protein kinases from these libraries using the following methods.
Expression:
( 1 ) Add 10 ml of cell culture medium to a Falcon centrifuge tube (50 ml).
( 2 ) Inoculate the culture into the medium along with bacteria expressing histidine-tagged protein kinase from 384 microtiter plates stored at -80°C.
( 3 ) After overnight incubation with vigorous shaking at 37°C, the cell cultures were transferred to 300 ml flasks containing 90 ml of pre-warmed 2YT medium containing 100 μg/ml ampicillin and 15 μg/ml kanamycin. Incubation was continued until the OD value reached 0.7.
( 4 ) To induce protein expression, IPTG was added at a final concentration of 1 mmol/L and incubated for 4 h at 37°C.
( 5 ) Transfer cell cultures to 50 ml Falcon centrifuge tubes (two tubes for each cell culture), centrifuge at 1900 g for 10 min, collect the cells, and immediately store at -80°C.
Purification of non-denatured proteins (the following steps were performed at 0-4°C):
( 1 ) Remove the precipitate kept frozen at -80°C and thaw on ice.
( 2 ) Lysed the cells with 0.5 ml of lysis solution containing 0.25 mg/ml lysozyme and 0.1 mmol/L PMSF.
( 3 ) Aggregate the lysis products and shear the DNA in the lysis products 3 times for 1 min each time in an ice bath using an ultrasonic homogenizer set at half power.
( 4 ) Transfer the lysate to a 1.5 ml centrifuge tube and centrifuge at 20,000 g at 4°C for 30 min.
( 5 ) Transfer the supernatant to a new centrifuge tube.
( 6 ) Add 250 μl of NiNTA-agarose and shake at 300 r/min at 4°C for 1 h to bind histidine-tagged proteins.
( 7 ) Transfer the mixture to a 1 ml polypropylene column.
( 8 ) Wash the column with 10-fold volume of non-denaturing wash solution.
( 9 ) Proteins are eluted in 4 elution steps, each with 0.5 ml of non-denaturing eluent.
( 10 ) Proteins are mixed with glycerol [final concentration 20% (V/V)] and stored at -80 °C (see Note 1). We recommend that during each step of centrifugation, lysis, washing, and elution, small samples be subjected to polyacrylamide gel ( e.g., 15% ) electrophoresis (Fig. 29-1) to test the efficiency of these purification steps. Protein concentration is determined using the Bradford assay [26] . 
3.2 Kinase Assays on Protein Microarrays It is recommended to test the kinase activity with a known substrate as a positive control prior to performing microarray experiments (see Notes 2, 3). It is also important to exclude the possibility that the kinase may contribute to the phosphorylation of the recombinant protein on 3' or 5' end tags, such as histidine tags (see Note 4).
Here, we present a method for signal analysis after kinase detection. We spotted sample proteins and controls (see Note 3) on FSAT slides (see Note 5) in a 10 X 10 spotting pattern (see Note 6) and repeated the spotting 4 times per sample. Spotting patterns for quantitative analysis have recently been reported [23].
Kinase assay:
( 1 ) The protein microarray (see Chapter 28 for construction method), which was sealed at 4°C for the first day of spotting, is washed with TBST for 1 h at room temperature with vigorous shaking (see Note 7).
( 2 ) The washed microarrays were blocked with 2% BSA/TBST for 1 h at room temperature.
( 3 ) Place the blocked chip on a TBST-soaked Whatman filter paper so that the chip will not dry out during the subsequent kinase reaction.
( 4 ) Incubate the microarray for 1 h at room temperature in 250 μl of kinase solution containing 13 μg/ml of CK2α and 25 μCi/ml of radiolabeled [ y33-P ] ATP (see Note 1). The incubation is performed under cover.
( 5 ) Wash 6 times with TBST for 30 min each time.
( 6 ) Finally, the chips are dried with a microtitre plate centrifuge (e.g., Eppendorf, Hamburg, Germany, Catalog No. 5810 R ) or blown dry manually and transferred to an X-ray cassette.
( 7 ) For signal detection with X-ray film, the film is placed on the chip and the X-ray cartridge is stored at -80°C for an appropriate period of time (see Note 8). The film is then developed in a darkroom. Alternatively, the film is exposed to an imaging plate and the signal is detected with a phosphor screen visualizer (see Note 8).
Figure 29-2 ( left) shows typical results from a CK2α phosphorylation screening experiment after exposure of chip-to-X-ray film, with four repetitive spot samples of several proteins giving different signals on the film.
To determine the localization of histidine-tagged recombinant proteins on the chip, we screened these proteins with an anti-RGS-His6 antibody described in this book (see Chapter 28). Almost all of the spot-sampled proteins were detected (about 95% ), as shown in Fig. 29-2 ( right). 
3.3 Selection of Potential Kinase Target Proteins
The results of two independent experiments in different ways for this kinase are needed for signaling analysis.
Selection criteria for potential kinase target proteins:
( 1 ) If all 4 spot sampling points of a sample in an experiment give an identical signal on X-ray film or phosphor screen visualizer (Fig. 29-2), then this protein is considered positive.
( 2 ) If a positive result is obtained in both experiments, the protein is considered a possible target protein.
For example, 21 possible CK2α target proteins were identified from close to 800 different barley proteins " 22 ". A method for screening potential target proteins with a threshold-based quantification system after microarray phosphorylation was recently reported.
3.4 Validation of potential phosphorylated target proteins
When using this microarray-based phosphorylation method as a screening tool for the identification of potential phosphorylated target proteins in vitro, these identified proteins are to be validated by other methods, both in vitro and in vivo, some of which have been mentioned in the introduction.
We used an imprinted phosphorylation assay to validate potential substrates ex vivo:
( 1 ) Proteins were separated by SDS-PAGE, e.g. 15% SDS-PAGE, and then transferred to a PVDF membrane. After transferring the membrane, the gel was stained with Caumas Brilliant Blue to examine the transfer efficiency of the proteins.
( 2 ) Perform the phosphorylation reaction on the blotting membrane using the reaction conditions based on microarray assay described in this book as well as the appropriate volume.
Figure 29-3 shows a classic example of an imprinted phosphorylation experiment. We validated 48 proteins identified as potential substrates with MAP kinase in two microarray experiments, and almost all of the proteins were confirmed, as shown in Figure 29-3. We used the same positive controls as in the microarray experiments: different concentrations of myelin basic protein ( MBP, an artificial substrate for MAP kinase). Negative control proteins were those detected in the microarray-based kinase assay but not yet identified as potential substrates (Fig. 29-3).
Other in vitro validation methods are phosphorylation assays in solution, SDS electrophoresis, and radioautography for phosphorylated proteins [8, 23], which was used by Fukunaga and Hunter to validate kinase substrates identified by phosphorylation screening of phage cDNA expression libraries.