Sample preparation
- Start a new culture at OD 0.02 from an exponentially growing culture when it reaches ~0.4 OD.
- At the desired OD, take your sample. Alternatively, a sample from a chemostat. Remember to record OD.
- Pull 1 mL of culture.
- Immediately spin the cells 13 K (max on tabletop centrifuge) for 2 minutes to pellet.
- Resuspend cells in lysis buffer.
- Final cell concentration will be 2 E6 cells/uL (use OD/cfu curve). This concentration is about right for MC4100-pSB4A3.I7101; adjust as necessary.
- Cell lysates can be frozen at this point (-20 C). Aliquot if desired.
Gel Preparation
see trick-tricine acrylamide gel protocol for pouring vertical acrylamide gels
- Boil samples for 10 minutes (use 95 C sand block).
- Spin at 13 K for 10 minutes.
- Mix samples and standards with 2X sample buffer (in PCR tubes) and boil for 10 minutes (95 C heat block). Spin down and load onto tric-tricine acrylamide gel (see JcBAcrylGel_protocol) immediately.
Run ~1 E7 cells per lane (5 uL of lysis sup’n and 5 uL of 2X sample buffer). 1 E7 cell/lane is good for pSB4A3.I7101. Adjust for higher or lower expression levels. Run GFPssrA standards (10 ng, 20 ng, 40 ng, 60 ng, 80 ng) in water (or neg. control sup’n) to a volume of 5 uL mixed with 5 uL 2X sample buffer. (20 ng/uL purified GFP aliquots at –80C; keep on ice until use) Run 3 uL Amersham Rainbow marker #755 as size marker.
Transfer and Incubations
- After running gel, cut away stacking gel, cut a corner to mark orientation, and soak in transfer buffer for 45 minutes.
- Cut PVDF and 3mm Whatman blotting paper to the correct size.
- Cut the corner on PVDF and wet it with methanol, then soak for 5 minutes in H20, followed by 10 minutes in transfer buffer. Wet 4 pieces of blotting paper in a transfer buffer.
- Set up transfer bottom to top: 2 pieces blotting paper, gel, PVDF, two pieces blotting paper. Be sure to preserve the orientation of the blot and avoid bubbles.
- Transfer 2 mA/cm^2 until transfer is complete (i.e. rainbow marker is fully transferred to PVDF membrane). For one 8.5 X 5 blot, this works out to 85 mA for about 2 hours.
- Block membrane for 1 hr with 5% milk/TBS-Tween (0.1 %). Use the lid of a pipette tip box and 15 mL fluid to cover the
- blot. (for two blots, use a large tip box and 25 mL fluid for all incubations/washes) Place blot on the shaker table for all incubations.
- Incubate overnight with 1:10000 dilution of anti-GFP in 2% milk/TBS-Tween (0.1%)
- Wash 3X in 15 mL TBS-Tween (0.1%) for 10 minutes.
- Incubate 30 minutes with 1:10000 dilution of ECF secondary antibody in TBS-Tween (0.1%)
- Wash 3X in 15 mL TBS-Tween (0.1%) for 10 minutes.
Analyzing Western
- Turn on Fluorimager 30 minutes before use. Put in 570 filters.
- Settings are PVDF 488/570 of 30, PMT = 500. Select an area to scan.
- Place 1 mL/gel of ECF substrate on a transparency. ECF substrate in the buffer is stored in 1 mL aliquots at –80C.
- Using forceps, lay the blot face down on top of the substrate (no bubbles) for about one minute (less if bands become visible). Transfer the blot face down to the fluorimeter plate.
- Insert the plate into the machine. Scan image. Remove the plate immediately.
- Remove the plate before shutting down the scanner. Leave software open if anyone is signed up within two hours.
- Clean the plate with DI and Kim wipes, then with ethanol from the glass bottle.
Quantification in ImageQuant
- Use a rectangle tool to draw objects around the band. Copy and paste objects so that all objects have the same area.
- It is not necessary to define a background in ImageQuant. Do volume report without background correction, and set the negative control equal to 0 ng/lane for your standard curve. Alternatively, make a new object to define the background and set the background on all lanes equal to “object ave” for this object. (analyze>>” background correction” to set background correction, then analyze>>” volume report”) (“local average” sets the background equal to the average pixel value of the object perimeter. This will be problematic if bands are not well separated.)
volume = (average pixel value – background value) * object area
- Double-click click report to make it an Excel file. Save images and Excel volume report.
- Use the standard curve to estimate ng gfp/lane for samples and calculate gfp/cell. The molecular weight of GFP-SsrA is about 28.3 kDa.
2X Tricine Sample Buffer
- 2 mL 4X Tris-Cl/SDS, pH 8.8
- 6 mL 40% glycerol (24% final)
- 0.8 g SDS (8% final)
- 0.31 g DTT (0.2 M final)
- 2 mg Coomassie blue G-250 (0.02% final) (used C. Blue G)
- to 10 mL with MilliQ H2O and mix
- aliquot 500 uL/tube and store at –20 C
4X Tris-Cl/SDS pH 8.8
- 91 g Tris
- dissolve in 300 mL H2O
- pH to 8.8 with 1N HCl (about 120 mL)
- to 500 mL with H2O
- filter 0.45 um
- add 2g SDS and store 4 C
Lysis Buffer (12.5 mM Tris pH 6.8, 4% SDS
- 1.25 mL 1M Tris (pH 8)
- to 80 mL with st. H2O
- pH if necessary
- to 100 mL with st. H2O
- add 4g SDS
TBS-Tween (0.1%)
- 100 mL 10X TBS
- 900 mL H2O
- 1 mL Tween (Polyoxyethylene sorbitan monolaurate)
10X TBS (500 mM Tris, 1.5 M NaCl)
- 150 mL 5M NaCl
- 250 mL 1M Tris, pH 7.5
- to 500 mL with H2O
1M Tris-Cl, pH 8 (or 7.5)
- 121 g tris base
- 700 mL MilliQ H2O
- to pH 8 with 6N HCl (about 100 mL)
- to 1 L with MilliQ H2O
- filter 0.45 um or autoclave (fluid, 20 psi, 250 F, 20 min)
Transfer Buffer (“Towbin Buffer”)
- 3 g Tris
- 14.4 g glycine
- 800 mL dI
- 200 mL methanol
note: anti-GFP is Assay Designs 915-059, rabbit (store one aliquot at 4 C to avoid freeze/thaw, rest at –20C) ECF Western Blotting kit is Amersham RPN5783 (rabbit) To connect to Bionet from fluorimeter: \\18.79.1.147\endy DNA-NET\username