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week number 30

▼2015-07-25 Yeast cell lysates using TCA

Cell sediment from 2-3 OD of cells was resuspended in 1 ml of cold water. Alternatively, resuspend cells (a match head) from a fresh plate in 1 ml water. Alternatively, just use 1 ml of your cells in the medium (does only work for synthetic media, rich media contain too much  TCA precipitable material). Add 150 µl of 1.85 M NaOH, 7.5% ß-mercaptoethanol to the resuspended cells. Incubate 10 min on ice. Than add 150 µl 55% TCA (w/v) to the resuspended cells. Incubate for 10 min on ice. Centrifuge at 4°C for 15 min in a microfuge (14000 rpm). Remove supernatant, centrifuge again and remove remaining supernatant. Resuspend precipitated proteins in 100-200 µl HU-buffer by vortexing and heating sample for 10-15 min at 65°C. Spin samples for 5-10 min at RT, full speed in Eppi centrifuge. Load sample (10-15 µl) onto SDS-PAGE.

HU-buffer (high-urea buffer):
8 M urea, 5% SDS, 200 mM NaHPO4 pH 6.8, 0.1 mM EDTA, bromphenol blue (according  to your taste), freeze at –20°C, before use, adjust to 15mg/ml DTT

▼2015-07-25 Western Blot

15µl Cell lysates were loaded on a 12% SDS Gel:
Resolivng Gel:
6.6 ml water
8 ml acrylamide mix 30
5 ml Tris 1.5 M pH 8.8
200µl 10% SDS
200µl 10% APS
20µl TEMED

Stacking Gel:
3.4 ml water
830 µl Acrylamid mix 30
630 µl Tris pH 6.8
50 µl 10% SDS
50µl 10% APS
5 µl TEMED

Western Blot
Cut pvdf membrane (9x6cm) and Blotting paper to the dimensions of the gel. One membrane and two pieces of extra thick filter paper per gel are needed
Activate membrane 5min in Methanol
Equilibrate gel in transfer buffer for 5min
Assembly:
1. Place a pre-soaked sheet of extra thick filter paper onto the platinum anode. Roll a pipet or test tube over the surface of the filter paper (like a rolling pin) to exclude all air bubbles.
2. Carefully place the equilibrated gel on top of the transfer membrane, aligning the gel on the center of the membrane. Transfer will be incomplete if any portion of the gel is outside the blotting media. Roll out all air bubbles.
3. Place the other sheet of pre-soaked filter paper on top of the gel, carefully removing air bubbles from between the gel and filter paper.
4. run Blot at 25 V for 1h

Prepare around 50ml 5% milk in TBT-T.
After blotting wash membrane three times with TBT-T for 5min
Block 1h or overnight with 5% milk, keep milk afterwards
Wash membrane three times, 5min with TBT-T
Prepare dilution of first antibody in 5 ml milk in a Falcon
Put membrane in the Falcon and let it roll at 4°C overnight
Wash membrane three times, 5min with TBT-T
Prepare dilution of second antibody in 5 ml milk in a Falcon
Put membrane in the Falcon and let it roll at 4°C for 1 h
Wash membrane three times, 5min with TBT-T
Prepare strain solution: 2.5 ml each in one Falcon
Put membrane in the Falcon and let it roll at 4°C for 5min
Pipet 50 µl H20 on a clear film, add membrane, pipet 50µl on the membrane and close clear film