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.
- 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.
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: \\18.104.22.168\endy DNA-NET\username