Endy:E. coli Western Blot

GFP Quantitative Western
JCB Protocol for plasmid based GFP; batch or chemostat culture; modification of Chris Farrell’s protocol

Sample preparation

 * Start new culture at OD 0.02 from an exponentially growing culture when it reaches ~0.4 OD.
 * At desired OD, take your sample. Alternatively, sample from chemostat.  Remember to record OD.
 * Pull 1 mL of culture.
 * Immediately spin the cells 13 K (max on table top 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 tric-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 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 correct size.
 * Cut corner on PVDF and wet with methanol, then soak 5 minutes in H20, followed by 10 minutes in transfer buffer. Wet 4 pieces of blotting paper in transfer buffer.
 * Set up transfer bottom to top: 2 pieces blotting paper, gel, PVDF, two pieces blotting paper. Be sure to preserve orientation of 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 blot. (for two blots, use a large tip box and 25 mL fluid for all incubations/washes) Place blot on 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 filter.
 * Settings are PVDF 488/570 df 30, PMT = 500.  Select area to scan.
 * Place 1 mL/gel of ECF substrate on a transparency. ECF substrate in buffer is stored in 1 mL aliquots at –80C.
 * Using forceps, lay blot face down on top of substrate (no bubbles) for about one minute (less if bands become visible). Transfer blot face down to fluorimager plate.
 * Insert plate into machine. Scan image.  Remove plate immediately.
 * Remove plate before shutting down scanner. Leave software open if anyone is signed up within two hours.
 * Clean plate with dI and kim wipes, then with ethanol from the glass bottle.

Quantification in ImageQuant
volume = (average pixel value – background value) * object area
 * Use rectangle tool to draw object around band. Copy and paste object 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 background and set 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 background equal to the average pixel value of the object perimeter.  This will be problematic if bands are not well separated.)
 * Double click report to make it an excel file. Save images and excel volume report.
 * Use 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 fluorimager: \\18.79.1.147\endy   DNA-NET\username