Mini protean gel electrophoresis system

Isolated pre-40S ribosome complexes were run on 12% polyacrylamide gels [12% (v/v) acrylamide/bis-acrylamide 37.5:1, 375 mM Tris/HCl (pH 8.8), 0.1% (w/v) SDS, 0.12% (v/v) APS, 0.012% TEMED (v/v)] and a stacking region [5% (v/v) acrylamide/bis-acrylamide 37.5:1, 125 mM Tris/HCl (pH 6.8), 0.1% (w/v) SDS, 0.08% (v/v) APS, 0.008% (v/v) TEMED]. Gels were resolved using a Mini-PROTEAN gel electrophoresis system (Biorad) in running Buffer [25 mM Tris/HCl, 192 mM Glycine and 0.1 % (w/v) SDS]. Gels were first fixed in 50% (v/v) Ethanol and 10% (v/v) Acetic Acid, then 5% (v/v) Ethanol and 1% (v/v) Acetic Acid for 30 min and 15 min respectively on a shaker at room temperature. The gels were then washed three times in ultrapure water for 5 min. Next, the gels were sensitised in 0.02% (w/v) Sodium Thiosulphate for 2 min and then washed again three times in ultrapure water (Milli-Q® Advantage A10 Water Purification System, Merck) for 30 s. The gels were stained in 0.1% (w/v) Silver Nitrate and 0.08% (v/v) formaldehyde in the dark for 20 min and washed again three times in ultrapure water for 20 seconds. The gels were finally developed in 2% (w/v) Sodium Carbonate, 0.04% (v/v) Formaldehyde and 0.0004% (w/v) Sodium Thiosulphate until protein bands were visible, then the reaction stopped using 5% (v/v) Acetic Acid. A photograph of the gel was then taken using a G:Box Chemi XX9 imager.

Total head extracts were used for semiquantitative densitometric Western blot. Briefly, 50 µg of protein were loaded in 12% Laemmli-SDS-PAGE gels (Biorad Mini protean gel electrophoresis system) and transferred to nitrocellulose membranes for 3 h at 250 mA. Membranes were blocked overnight at 4ºC temperature with PBS 0.1% tween (PBST) supplemented with 10% milk. Membranes were then incubated at 4 °C overnight with antiTH (Inmunostar 1:1000) in 5% milk PBST, washed 3 times with PBST and incubated at room temperature for 1 h with KPL HPRT goat-antimouse (Sera Care) in 5% milk PBST. Blots were then washed 3 times in PBST, and bands were detected using the SuperSignal West pico Chemiluminiscence Substrate (ThermoFisher) according to manufacturer’s instructions. Blot signals were digitalized and quantified using ImageJ. Mouse monoclonal anti-actin (DHB, 1:3000) was used as loading control.

We performed sodium dodecyl sulfate (SDS) polyAcrylamide gel electrophoresis (PAGE) using 12% resolving and 5% stacking gels for separating proteins. We followed the Laemmli’s method
^{9 (link)} for gel electrophoresis. The samples were mixed with equal volume of gel loading buffer and heated at 95°C in dry heating bath for 2 mins. The electrophoresis process was run with 90 V for first 10 mins and then run at 150 V with Biorad mini protean gel electrophoresis system. After complete run the gel was stained with Coomassie Brilliant Blue. We have used protein marker (10kD to 250 kD) from GCC biotech (Pre-stained protein marker GCR-P4B) for determination of molecular weight. We imaged the gels in Biorad gel documentation system. Acrylamide, bis Acrylamide, Tris and TEMED (T9281) are from Sigma Aldrich. Coomassie Brilliant Blue R250 (93473) and Ammonium per sulphate (28575) was from SRL (Sisco Research Laboratories).