Perfection v500 photo flatbed scanner

The 26S proteasomes were co-purified with polyubiquitin conjugates on ^GstUBD beads followed by incubation at 37°C for 7 minutes, centrifugation at 12,000 rpm/min for 1 minute and immediate processing of the collected supernatants for EM. Briefly, undiluted 5 µl aliquots were applied to plasma-discharge treated carbon-coated support grids, adsorbed for approximately 30 sec at room temperature, washed with water, and stained with 2% uranyl acetate (unadjusted pH) for approximately 30 sec before blotting and drying. Samples were examined with a JEOL 1200EXII electron microscope, using magnifications of 40,000 or 60,000 to record images on Kodak SO-163 film at either high-dose or minimal-dose conditions. Films displaying several proteasomes per field were scanned using an Epson Perfection V500 Photo flatbed scanner, using resolutions of 846 or 1270 dpi to yield a pixel size of 5Å. All particles recognized as 20S end and side views, and 26S side views, as well as rings and circles of reproducible dimensions, were selected from these scanned images with the EMAN [62] (link) routine Boxer. After eliminating particles that were recognizable but poorly stained, the selected particles were subjected to multiple rounds of reference-free alignment and averaged using SPIDER [63] .

DNA (≥10 µg) was digested overnight with appropriate restriction enzymes and run on a 0.8% agarose gel to separate fragments. Gels were subject to depurination and neutralisation before transferring DNA to Hybond-XL membrane (RPN203S, GE Healthcare Amersham) overnight by capillary action. The membrane was then washed and dried at 80 °C for 2 h. αP32–dCTP probes were labelled using Prime-It® II Random Primer Labelling Kit (300385, Agilent) and purified with Illustra ProbeQuant™ G-50 Micro columns (28-9034-08, GE Healthcare Amersham). Following pre-hybe for 1–2 h in Rapid-hyb™ Buffer (RPN1636, GE Healthcare Amersham), the membrane was hybridised with the probe for at least 5 h, washed, exposed to Fuji RX X-ray film and the film scanned on an Epson Perfection V500 Photo flatbed scanner.

To determine the linear dimensions of 9-day-old plantlets, they were laid out individually on the glass of an Epson Perfection V500 Photo flatbed scanner (Epson, Suwa, Nagano, Japan) and scanned at a resolution of 800 dpi. The public domain software ImageJ v.1.49 (NIH; http://rsb.info.nih.gov/ij, accessed on 30 January 2021) was used to measure the lengths of the plantlets (root, hypocotyl, cotyledons, whole plantlet) to an accuracy of 0.1 mm.