Model 583

For the purpose of pulse labeling of newly translated proteins, 50 isolated islets were incubated in complete RPMI media without cysteine and methionine (MP Biomedicals, #SKU 091646454) for 1 h. Subsequently media was supplemented with 250 μCi of [³⁵S]-cysteine/methionine mixture (PerkinElmer, NEG772002MC) and islets were incubated under normal conditions for 30 min. Islets were then lysed and proteins separated by SDS-gel electrophoresis as described above. Gels were fixed for 30 min in 50% (v/v) ethanol in water with 10% (v/v) acetic acid, dried in gel dryer (Bio-Rad model 583) and then exposed to storage phosphor screen (GE Healthcare) overnight. Screens were imaged and digitized using Typhoon FLA 9000 biomolecular imager (GE Healthcare). Protein bands intensity was quantified with Adobe Photoshop software version 7.

The H53 parent strain carrying the vector control pTA963 and the ΔhvpssA mutant strain carrying either the hvpssA expression plasmid pFH39 or the vector control pTA963 were grown in 5 ml liquid CA medium. Upon reaching mid-log phase (OD₆₀₀, ∼0.5), 20 μl of each culture was transferred into 1 ml of fresh liquid CA medium supplemented with [¹⁴C]mevalonic acid (resuspended in ethanol) at a final concentration of 1 μCi/ml. Haloferax volcanii cultures were harvested after reaching mid-log phase, and proteins were precipitated from 1-ml cultures with 10% trichloroacetic acid (TCA), followed by a delipidation step to remove noncovalently linked lipid, as described previously (44 (link), 45 (link)). The delipidated proteins were separated by 7% Tris-acetate (TA) LDS-PAGE gels. For analysis of the samples, the gel was dried onto blotting paper using a gel dryer (model 583; Bio-Rad), exposed to a phosphor screen (Molecular Dynamics) for 3 weeks, and analyzed using a Typhoon imager (Amersham Biosciences).

750 ng– 1 μg of XhoI digested genomic DNA was subjected to agarose gel electrophoresis (0.7% agarose) for 16 hours at 60 V. The gel was placed on a double-layer of Whatman paper with a piece of plastic wrap over top and mounted on a Bio-rad (model 583) gel drier. Drying was carried out for ~20 minutes at room temperature. The dried gel was sealed in a plastic bag and hybridized with at least 1 million CPMs of γ-ATP32 end-labeled oligonucleotide 5’-CCCACCCACCACACACACCCACACCC-3’ in in-gel hybridization buffer overnight in a 37°C water bath. After removal of excess hybridization buffer, the gel was washed 3–4 times for 30 minutes in 0.25X SSC at room temperature with agitation, sealed in a bag, and exposed to Amersham Hyperfilm MS or phosphor cassette. After, the in-gel was subjected to a denaturing Southern blot in order to visualize the total telomeric DNA. The Southern blot was performed as follows: 10 minutes in 0.25M HCl, 45 minutes in Southern denaturing solution (1.5M NaCl, 0.5M NaOH), 5 minutes 0.4M NaOH. It was then transferred to a Hybond-XL nylon membrane. After transfer, the membrane was prehybridized for 1 hour in Church buffer and then hybridized overnight at 65°C with a radiolabeled probe that hybridizes to telomeric repeats. The membrane was washed for 20 minutes at room temperature in 2X SSC and exposed on a phosphor cassette.