Experiments on the construction of plant protein microarrays and the study of antigen-antibody interactions
The completion of the Arabidopsis and rice genome sequencing projects has greatly supported the application of proteomics methods such as protein microarray technology. In this chapter, we present a method for the construction of plant protein microarrays and its application to the study of specificity and cross-reactivity of monoclonal antibodies or polyclonal sera. The source for this experiment is the "Guide to Plant Proteomics Experiments" [French] H. Tillemment, M. Zivi, C. Damerweil, V. Mitchen, eds.
Operation method
Construction of plant protein microarrays and study of antigen-antibody interactions
Materials and Instruments
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PP Buffer Denaturing Lysis Buffer
Microtitre plate
We used an E. coli cDNA expression clone constructed in the PQE30 expression vector that expresses an N-terminal RGS-His6 tag to produce recombinant plant proteins. Since it is beyond the scope of this section, we will not describe here in detail the process of generating such expression clones, but only briefly summarize it. We may be able to obtain the plant expression clones described above from the following cDNA expression libraries: a barley expression library constructed in the expression vector PQE30NST ( Retrieval No. AF074376 ) and an Arabidopsis expression library constructed in the pQE30NAST attB vector (Retrieval No. AF386205 ). These expression libraries have been used to produce recombinant proteins with an intact N-terminus using deoxythymidine deoxyoligonucleotides as primers according to the method of Konrad Buessow's group [ 34~36]. We used a full-length Arabidopsis clone as an alternative source of plant protein expression clones, and this full-length Arabidopsis clone was obtained by direct cloning in a readable frame using gene-specific primers for restriction-dependent cloning, or recombination-dependent cloning using the GATEWAY cloning technique.
To express plant proteins in high throughput and on a small scale from these sources mentioned above, we used the following approach.
Protein expression
( 1 ) A 96-well deep well plate with a well volume of 2 ml was filled with 100 μl of 2YT medium supplemented with 2% glucose, 100 μg/ml ampicillin and 15 μg/ml kanamycin.
( 2 ) Recombinant clonal cultures are inoculated from 384- or 96-well microtiter plates previously stored at -80°C using a 96-pin replicator.
( 3 ) Cultures were grown at 37°C for 16 h with vigorous shaking (320 r/min), then 900 μl of pre-warmed medium (1X SB medium, 1X PP buffer with 100 μg/ml ampicillin, 15 μg/ml kanamycin, and 20 μg/ml thiamine) was added, and incubation was continued for another two hours.
( 4 ) To induce protein expression, IPTG at a final concentration of 1 mmol/L was added and incubation was continued for 4~5 h. The incubation was then continued for 2 h. The incubation was then continued for 2 h.
( 5 ) Centrifuge the cells at 1500 g for 10 min at 4°C, collect the cells, and store the sediment at -80°C.
( 6 ) Remove a portion of the lysate from the harvested cells and examine the protein expression efficiency on a 15% polyacrylamide gel (Figure 28-1). 
Protein Purification
( 1 ) First, add 150 μl of denaturing lysis buffer to the thawed precipitate, dissolve the precipitate by vigorous vortexing and oscillation, and incubate for 30 min at room temperature with shaking (about 650 r/min) (see Note 2).
( 2 ) Lysed cells were centrifuged at 1900 g. The supernatant was transferred to a 96-well filter. The supernatant is transferred to a 96-well filter plate (see Note 3) and quickly aspirated onto a fresh plate using a vacuummanifold (see Notes 4 and 5).
( 3 ) Next, add 30 μl NiNTA-agarose (1 : 2 dilution in lysate) to each well, seal with tape, and shake for 1 h at 300 r/min at room temperature to bind the histidine marker to the protein.
( 4 ) Resuspend and wash the agarose microspheres with 100 μl of washing buffer, shake for 5 min, repeat 3 times, and then transfer the supernatant to a vacuum manifold (see Note 5).
( 5 ) Finally, the proteins are eluted from the agarose microspheres by static incubation of the agarose microspheres with 80 μl of eluent for 20 min, and the eluent is filtered and transferred to a fresh 96-well plate (see Note 6).
( 6 ) Store the protein at 4°C.
( 7 ) Purified proteins can be separated, e.g., with a 15% polyacrylamide gel (Fig. 28-2). Protein concentration is determined using the Bradford protein assay.
Using the method described above, we obtained purified proteins with an average concentration of approximately 100 μg/ml (data not shown).
3.2 Construction of plant protein microarrays
We used the FAST spot sample substrate as a surface for constructing plant protein microarrays for antibody analysis or protein phosphorylation studies (see Chapter 29). 
( 1 ) Before constructing the microarray, transfer 20 μl of purified plant protein and control (see Note 8) to a 384-well plate.
( 2 ) The FAST chip substrate was placed on a QArmy chip detection system with a humidity controller (65%-70%) and 16 blunt-tipped stainless steels with a tip diameter of 150 μm and a needle spacing of 4.5 mm. a spot depth of 120 μm was used.
( 3 ) Each sample was injected once, and the volume of each spot was 0.6 nL.
( 4 ) After each sample count, the sampler was cleaned with double-distilled water (6 s), blown dry by a hot air blower (2 s), and then cleaned with 80% ethanol (6 s), and blown dry again to prevent cross-contamination.
( 5 ) Protein microarrays are stored at 4°C in an airtight cassette.
Proteins can be spotted in different template forms, such as 4 X 4, 8 X 8, 10 X 10, etc. (see Note 9). Similarly, proteins can be spotted repeatedly, e.g. 2, 4 or more times. For the study of antibody-antigen interactions, it is recommended that two identical regions be spotted twice in the horizontal direction (see Note 10).
Plant Protein Microarrays constructed in this way can be used for antibody analysis studies and provide reproducible results for at least 3 weeks when stored at 4°C. In addition, this plant protein microarray can also be used for protein kinase phosphorylation studies [ 8, 33], which are described in detail in this book (see Chapter 29).
To detect recombinant plant proteins immobilized on the ProteinChip substrate, the chip substrate is incubated in anti-RGS-His6 antibody as follows (see Section 28.3.3 1). Figure 28-3 illustrates an example of a chip containing 96 Arabidopsis proteins expressed and purified from a cDNA expression library [33]. The proteins are repeatedly spotted in a 4 X 4 pattern in two identical regions, horizontally, and all recombinant proteins emit a single signal indicating the efficiency of the protein expression and purification, and also that the amount of sample transferred to the chip is sufficient for detection. the tested recombinant proteins on the chip (Figure 28-3). 
3.3 Antibody Screening on Protein Chips
1. Monoclonal Antibody Screening on FAST Spotting Substrates
( 1 ) Plant protein microarrays were blocked with 2% BSA/TBST for 1 h at room temperature.
( 2 ) Mouse anti-RGS-His6 ( 1 : 2000 dilution) or plant-specific antibodies (e.g. rat anti-TCP1 antibody; 1 : 1000 dilution) are diluted in the blocking solution (see Note 11).
( 3 ) Rinse twice with TBST for 10 min each time.
( 4 ) Incubate the chip substrate for 1 h at room temperature with the corresponding Cy3-labeled secondary antibodies (rabbit anti-mouse IgG with anti-RGS-His6-antibody; rabbit anti-rat IgG with anti-TCP1 antibody), which are diluted at a ratio of 1:800 in the blocking solution (see Note 13 and Note 14).
( 5 ) Rinse 3 times with TBST for 30 min each time (see Note 15).
( 6 ) Prior to scanning, we shook the chip dry with a microplate centrifuge (Eppendorf, Hamburg, Germany, catalog no. 5810R) or blew it dry manually.
( 7 ) Signals were detected with a 428Arrayscanner scanner or a ScanArray 4000 scanner.
All antibody incubation steps are performed in 200 μl wells under cover slip.
In order to exclude non-specific binding of secondary antibodies, we performed a set of parallel experiments, i.e., incubation of the protein microarrays in a blocking solution without primary antibodies followed by incubation with the corresponding secondary antibodies, including the rinsing step described above.
Figure 28-4 shows a fluorescence image of an Arabidopsis protein microarray after incubation with a monoclonal anti-TCP1 antibody.96 Arabidopsis proteins obtained by full-length cDNA expression cloning were adsorbed on FAST spotting substrates (4 X 4 spotting pattern, 2 repetitions of spotting horizontally in two identical regions). This figure shows that the anti-TCP1 antibody specifically recognizes only the TCp1 protein spots and does not cross-react with other adsorbed Arabidopsis proteins (Figure 28-4). 
2. Screening of Polyclonal Antibodies on FAST Spotting Substrates
( 1 ) FAST spotting slides were blocked in fish gel (10% dissolved in TBST) for 1 h at room temperature.
( 2 ) Dilute rabbit serum in the blocking solution (e.g. anti-DOF11 at a dilution ratio of 1 : 1000, anti-MYB6 serum at a dilution ratio of 1 : 500), and then incubate the microarray spotting substrate with it for 1 h at room temperature.
( 3 ) Rinse with TBST twice for 10 min each time.
( 4 ) Continue incubation of the spotting substrate for 1 h at room temperature with goat anti-rabbit IgG-Cy3 ligand, which is diluted at a ratio of 1 : 800 in the blocking solution (see Note 13).
( 5 ) Rinse 3 times with TBST for 30 min each time.
( 6 ) Before scanning, we dried the microarrays with a microplate centrifuge (Eppendorf, Hamburg, Germany, catalog no. 5810 R ) or blew them out manually.
( 7 ) Signals were detected with a 428Arrayscanner scanner or a ScanArray 4000 scanner. All antibody incubation steps are performed in 200 μl wells under cover slip.
3.4 Image Analysis
Point average intensity data (minus background) can be obtained with GenePixPro 4.0; for further comparison, intensity averages can be obtained by counting duplicate points.