Screening PETAL array with proteomic antigens

1.      Preparation of proteomic antigens. Proteomic antigen samples, such as whole cell lysate, cell membrane extraction, organelle extraction, and etal., are prepared according to laboratory protocols. For example, 1x RIPA buffer may be used to lysis animal cells. The lysis buffer shouldn’t contain reagents with free amino-group (-NH2), like Tris, Gly, since the sample are labeled with NHS-biotin, using the amino-groups on Lys. If there is –NH2 in the buffer, additional dialysis process is required. Buffer for dialysis is suggested as 1x PBS.

2.      Qualification of proteomic antigens. Proteomic antigens are measured for total protein concentration, and qualified on SDS-PAGE for protein integrity.

3.      Biotinylation of proteomic antigens. Proteomic antigens are labeled with Thermo Fisher Sulfo-NHS-LC-Biotin (#21336) following the instruction. For example, 200 ug proteomic antigen dialyzed in 1 ml 1x PBS, is mixed with 20 ug fresh prepared biotin solution (ratio of protein:biotin=10:1). After 30 min incubation at room temperature, 0.1 ml 0.1 M Tris, pH8.0 is added into the mixture to stop the reaction, and block the remaining active biotin. Usually a parallel biotinylation reaction control is performed for a whole cell lysate from 293T cells as control.

4.      Qualification of biotinylated proteomic samples. Biotinylated proteomic antigens are qualified following procedure same as western blotting. For example, 0.1 ug, 0.01 ug, 0.001 ug biotinylated proteomic antigens are separated on SDS-PAGE, and transferred to NC membrane. After blocking and washing steps, the biotin groups are detected with SA-HRP. A well biotinylation proteomic antigen will show evenly distributed biotinylated protein bands, and in intensities comparable to control samples.

5.      Blocking of PETAL arrays. One PETAL array slide (2x4 cm) is blocked with 10ml 10% BSA, 1x PBS, pH7.4, at 4 C overnight.

6.      Incubation of proteomic samples with PETAL arrays. An optimized amount of proteomic antigen used for screen PETAL array might varies, depending on the complexity of proteomic antigen. For examples, the amount used for proteomic antigen with hundreds proteins could be less than antigen with thousands proteins, and more samples may be used for proteomic antigen with high abundant proteins. For a general proteomic antigen such as whole cell lysate, 10 ug total protein per array is recommended. Thus 10 ug biotinylated total protein is diluted into 10 ml 1x PBS containing 10% BSA, and added to PETAL array replacing the blocking buffer. Then the arrays are gently shaking on a shaker for 3 hrs at 4 C.

7.      Washing. After incubation, the arrays are washed with 50 ml 1x PBS per array for 10 min. Repeat washing steps for 9 x.

8.      Incubation with fluorescent labeled SA. Fluorescent dye is selected according to the scanner requirement. For example, Streptavidin-Cy3 (Sigma, #S6402) is used for GenePix 4200A Microarray Scanner (Molecular Devices, LLC.) with 532 nm laser. After washing step, SA-Cy3 is diluted into 10 ml 1x PBS per array at 1:5000 ratio, and incubated the array for another 45 min at room temperature.

9.      Washing. Wash the array with 50 ml 1x PBS per array for 10 min. Repeat wash for 3x.

10.   Rinse and drying. Briefly rinse the array with 50 ml H2O per slide to remove residual salts for 3x. Then centrifuge the arrays on a slide holder at 1,000 rpm for 1 min to remove H2O.

11.   Scan and analyze the array at an array scanner, ie.: GenePix 4200A Microarray Scanner.

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