Ni-NTA Plate

Preparation of Ni2+-NTA coated microtiter plates

The Ni2+-NTA coated microtiter plate was prepared as described previously [27], except an ordinary ELISA carboxylated polystyrene microplate (polystyrene high BIND microplate, Corning life science, USA) was used instead of a plate coated with maleic anhydride (Pierce, USA) as the later is 20 times more expensive. Briefly, a microtiter plate was coated with Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (Sigma Aldrich, USA) overnight and washed 3 times with 0.05% Tween 20 before blocking with 3% BSA in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20. The coated plate was washed with a series of buffers including 1) 50 mM Tris-HCl, pH 7.5, 500 mM imidazole, 0.05% Tween 20 2) 0.05% Tween 20 3) 100 mM EDTA, pH 8.0 and 4) 0.05% Tween before charging with 10 mM NiSO4 for 20 minutes at room temperature. After washing with 0.05% Tween 20 and then with 50 mM Tris HCl, 500 mM NaCl, pH 7.5, the plate was then ready to be used to capture (His)6-containing proteins.

Surface functionalization of GOPTS & PDITC with Ni-chelated NTA

AB-NTA, Aminobutyl-NTA, N-(5-Amino-1-carboxypentyl)iminodiacetic acid Aminobutyl-NTA (AB-NTA) free acid replaces AB-NTA (disodium salt form; former Product Code: A296-10). The new free acid form is less hygroscopic than the disodium salt form. Dissolving the new acid form in a buffer involves the same procedure as dissolving disodium salt in a buffer. The new acid form can also be dissolved in water with sonication (up to 3%). Aminobutyl-NTA was first used by Dr. Hochuli in 1987 to purify recombinant proteins (the “Histag Etechnique). Since then, the AB=NTA has become an indispensable tool for immobilizing proteins with high specificity on solid surfaces, such as glass or the gold electrode. A solid surface is modified by AB-NTA and bio-functionalized via Ni (II) with a genetically expressed protein bearing a hexahistidine extension at its terminus. The His-tag technique has become increasingly important, particularl in surface plasmon resonance and structural analyses of proteins by x-ray interference. Using the His-tag technique, Dr. Noji was able to directly observe the rotation of F1-ATPase by fluorescence microscope.

Nα,Nα-Bis(carboxymethyl)-L-lysine

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/3/c9609dat.pdf

prepare 80 µL 457 mM AB-NTA, 1 M NaHCO3 solution per slide sandwich

use 0.06 g Nα,Nα-Bis(carboxymethyl)-L-lysine and 0.042 g NaHCO3 in 500 µL aqua dest

use 0.096 g Nα,Nα-Bis(carboxymethyl)-L-lysine and 0.067 g NaHCO3 in 800 µL aqua dest

use 0.108 g Nα,Nα-Bis(carboxymethyl)-L-lysine and 0.076 g NaHCO3 in 900 µL aqua dest

put 80 µL on one slide and put the other one upon → Make sure the upper sides were laid together

incubate o/n

prepare blocking solutions:

blocking solution for GOPTS: 10mg/ml BSA + 0.5% ethanolamine in PBS

blocking solution for APTES/PDITC:5mg/ml BSA + 5% ethanolamine in PBS

transfer slides into blocking solution for 30 min-60 min

2x wash slides for 10 min in PBS (shake it)

prepare 20 mL 1 % (w/v) NiSO4 solution per 5 slides (= 0.2 g/20 mL)

incubate slides in slide holder with nickel sulfate for 1 h

2x wash slides for 10 min in PBS (shake it)

prepare 1/10 PBS-dilution in aqua dest and check for pH (should be 7-8)

wash slides in diluted PBS-solution

Dry slides with wafer gun

Spotting and washing

spot using 11-spot mask

incubate o/n @ 4°C

remove protein solution with pipette

incubate twice 5 min with 10 mg/mL BSA solution in the wells

(flush slide with PBS)transfer slide into PBS solution and remove mask

wash 10 min in PBS

wash 10 min in PBS (with 20 mM Imidazol, 27.2 mg in 20 mL PBS)

wash 10 min in 1/10 diluted PBS

dry with wafer gun

measure fluorescence

incubate for 45 min in Strep-Cy5 solution (2µg/ml)+ 5-10mg/ml BSA

2x wash 10 min in PBS (shake it)

wash 10 min in 1/10 diluted PBS

dry with wafer gun

measure Cy5-fluorescence in microarray scanner