Lectin Microarray

 Prepare 1.5 mL micro-centrifuge tubes labeled as BSA in a tube rack and add 240 μL PBS to one of the BSA tubes.

 Add 10 μL of 2 μg/mL BSA solution to the BSA tube for preparing 80 μg/mL BSA solution, and mix it.

 Place the tube of BSA 80 μg/mL and a tube of the glycoprotein sample prepared from protocols 13.1.1 to 13.1.3 (above).

 Prepare in a 96 well-microplate a serial dilution series of BSA solution starting from a concentration of 80 μg/mL (well volume, 100 μL), by diluting it by a ratio of 1/2, down to 1/128 of the initial concentration.

 Prepare the same dilution series in the microplate for the glycoprotein sample.

 Mix Micro BCA Reagent A (MA) 2.5 mL, Micro BCA Reagent B (MB) 2.4 mL and Micro BCA Reagent C (MC) 100 μL in a 15 mL tube (hereinafter WR means a Working Reagent prepared in this way).

 Add 100 μL WR to all the wells of the microplate.

 Seal all wells with a plate seal.

 Incubate at 37°C for 2 h.

 Remove the plate seal.

 Measure the absorbance at 562 nm in a microplate reader.

 Dilute samples to 50 μg/mL by adding PBS based on the result of the Micro BCA Protein Assay. In the case of whole cell lysates, use PBS-T in place of PBS.

 Add 20 μL of each diluted sample into a tube containing Cy3 amount which is used for labeling of 100 μg proteins.

 Thoroughly mix each sample using a pipette.

 Centrifuge to spin down any fluids on the tube sidewall.

 Place the tubes in a container shielded from the light and incubate for 1 h at 25°C.

 Prepare Zeba spin columns.

 Remove the column’s bottom plugs and loosen caps (do not remove the caps).

 Place the column in a 2.0 mL micro-centrifuge tube.

 Centrifuge at 1,500 × g for 1 min to remove storage solution.

 Mark on the side of the column at where the compacted resin is slanted upward. Place the column in the micro-centrifuge with the mark facing outward in all the subsequent centrifugation steps.

 Add 300 μg for 1 min to remove buffer.

 Repeat Step 4–6 twice (3 times in total), discarding buffer from the collection tube.

 Place the column in a new collection tube, remove the cap and apply all of the sample (20 μL) to the top of the compact resin bed.

 Apply 25 μL of TBS to the top of the gel bed after the sample has fully absorbed.

 Centrifuge at 1,500 × g for 2 min to collect the sample.

 Discard desalting columns after use.

 Measure each volume of the Cy3-labeled samples with a micro-pipette.

 Prepare a total volume of 1 mL by adding GP Biosciences’s probing solution. The protein concentration becomes 1 μg/mL as 1 μg protein is eluted.

 Serial dilute each sample eluate to 500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.25 ng/mL, 15.625 ng/mL using probing solution.

 Take out the LecChips (stored at −20°C) and place in an incubation box.

 Apply 100 μL Probing Solution with an 8 channel multi-pipette to well of the LecChips.

 Throw away Probing Solution and place a LecChip face down on a paper (e.g., Kimtex Quarterfold) to blot Probing Solution from the wells.

 Before drying up the wells, apply 100 μL Probing Solution to the wells.

 Repeat the above step 6 and step 7 (Probing Solution washing 3 times in total).

 Throw away Probing Solution and apply 100 μL of these samples to each well of the LecChips with a pipette.

 Incubate the LecChips in an incubation box at 25°C for at least 16 h with shaking at a low speed.

 Scan the LecChips with a GlycoStation^TM Reader following the recommended reader settings and conditions. In order to detect very weak signals while avoiding saturation of strong signals, it is recommended that you take additional scans while adjusting the gain and the exposure time around the recommended condition.

 Data is analyzed with Array-Pro^TM Analyzer Ver.4.5. The net intensity for each lectin spot is calculated by subtracting the background from the signal intensity. The digitized data is displayed differentially with GlycoStation^TM Tools.

 The latest GlycoStation^TM Tools Pro Suite covers the function of Array-Pro^TM Analyzer Ver.4.5.