Current Protocols in Chemical Biology 2010 (Volume 2) by Adam P. Arkin

By Adam P. Arkin

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These controls will provide standards for scanning and aid in aligning images during data processing. 3. Place 15 μl of solution into the appropriate well(s) of a 384-well V-bottom sample plate with lid. Depending on the number of samples and setup of the sample plate, multiple sample plates may be needed. Glycan Microarrays The maximum number of spots that can be printed within a well depends on a number of parameters, including the spot size, the pitch, the location and layout of pins, and the type 38 Volume 2 Current Protocols in Chemical Biology A B 2 ϫ 2 pin configuration Figure 1 Coordination of slide layout, configuration of sample plate, and pin positioning.

Pooled serum from multiple donors can provide a reference when analyzing serum samples. To minimize freeze-thaw cycles for the reference sample, divide a single 44 Volume 2 Current Protocols in Chemical Biology Table 2 Recommended Buffers and Starting Dilutions of Samples for Incubation on Glycan Microarray Sample type Incubation buffera Concentration Lectin 1% BSA in PBST 1-50 μg/ml Room temperature for 2 hr Monoclonal antibody 3% BSA in PBST 1-50 μg/ml 37◦ C while gently shaken for 2-4 hr Serum antibodies 3% BSA in PBST 1:50 to 1:200 Conditions 37◦ C while gently shaken for 4 hr a PBST: PBST: see recipe for PBST array wash buffer in Reagents and Solutions.

14. Resuspend beads in 20 μl 1× RIPA buffer containing 1× protease inhibitor, 1× phosphatase inhibitor, and 1× sample buffer for SDS-PAGE. 15. 5 min. 16. Load the samples onto an SDS-PAGE gel and proceed with immunoblotting as described in steps 6 to 8 of Support Protocol 1, using thiophosphate ester rabbit monoclonal antibody. PREPARATION OF A THIOPHOSPHORYLATED POSITIVE CONTROL PEPTIDE AND PROTEIN The two control samples, a thiophosphorylated peptide and protein, can be prepared from easily purchased materials.

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