Endy:E. coli Western Blot

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Revision as of 22:43, 25 July 2005

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.
• 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.

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 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

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