Endy:E. coli Western Blot
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GFP Quantitative Western
JCB Protocol for plasmid based GFP; batch or chemostat culture modification of Chris Farrell’s protocol
- Start new culture at OD 0.02 from an exponentially growing culture when it reaches ~0.4 OD.
- Around OD 1.0, 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). Cell lysates can be frozen at this point (-20 C). Aliquot if desired.
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.
- Run 1 E7 cells per lane (5 uL of lysis sup’n and 5 uL of 2X sample buffer).
- 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.
(20.8 ng/uL GFP-SsrA aliquots at –80C; keep on ice until use)
- Run 3 uL Amersham Rainbow marker #755 as size marker (gfp runs w/ orange band).
- 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.
[1 E7 cell/lane is good for pSB4A3.I7101. Adjust for higher or lower expression levels]
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 (about 8.5cm X 5cm for PVDF, blotting paper can be a bit larger)
- 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.
- 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 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. Return plates to cabinet across hall.
Quantification in ImageQuant
- 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.
- Double click report to make it an excel file and save images and excel.
- 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.)
volume = (average pixel value – background value) * object area
- Use standards 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
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: \\184.108.40.206\endy DNA-NET\username