title: NCI-60 cell line Reverse Phase Protein Lysate Array_modified
status: Public on Feb 01 2007
contact.city: Bethesda
contact.first: Uma
contact.middle: T
contact.last: Shankavaram
contact.email: uma@mail.nih.gov
contact.state: MD
contact.address: 9000 Rockville Pike
contact.department: NCI
contact.country: USA
contact.institute: NIH
contact.phone: 301-496-6718
organism: Homo sapiens
description: 640 spots (plus eight spatial registration marks for use in image processing) on a 21 x 35 mm area of nitocellulose membrane.
manufacture_protocol: Arrays were prepared on nitrocellulose-coated galss slides (FAST Slides, Schleicher & Schuell) by using a pin-in-ring format GMS 417 arrayer (Affymetrix, Santa Clara,CA) with four 500 um diameter pins. Because the cell lysates were viscous, we used pin-in-ring format to avoid clogging of quills. Ten two-fold serial dilutions were made from each lysate. Four 384-well microtiter plates (Genetix, New Milton, Hamphisire, UK) were used to array 640 spots (plus eight spatial registration marks for use in image processing) on a 21x 35 mm area of nitocellulose membrane. The first dilution (four-fold) was made with buffer containing 5M urea, 2% Pharmalyte, pH 8-10.5, and 65 mM DTT. Hence, only the lysate concentration changed along each dilution series. The urea concentration was kept at 6M and CHAPS concentration 2%, to keep proteins in their denatured form. We could remove lysates repeatedly from -80 degreeC storage for use without heating. To avoid evaporation in the microtiter plate during spotting, we kept the humidity in the array chamer at 70-90% with a Vicks ultrasonic humidifier. Arraying was completeed for each microtiter plate within 70 min. Arrays were produced in batches of 20, and the occassional low-quality array (e.g. with many spots dropouts) was discarded.