Note: anti-GFP is Assay Designs 915-059, rabbit (store one aliquot at 4 C to avoid freeze/thaw, rest at –20C)ĮCF Western Blotting kit is Amersham RPN5783 (rabbit) filter 0.45 um or autoclave (fluid, 20 psi, 250 F, 20 min).1 mL Tween (Polyoxyethylene sorbitan monolaurate).Lysis Buffer (12.5 mM Tris pH 6.8, 4% SDS 2 mg Coomassie blue G-250 (0.02% final) (used C.The molecular weight of GFP-SsrA is about 28.3 kDa. Use standard curve to estimate ng gfp/lane for samples and calculate gfp/cell.Double click report to make it an excel file.Volume = (average pixel value – background value) * object area This will be problematic if bands are not well separated.) (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. Alternatively, make a new object to define background and set background on all lanes equal to “object ave” for this object. Do volume report without background correction, and set the negative control equal to 0 ng/lane for your standard curve. It is not necessary to define a background in ImageQuant.Copy and paste object so that all objects have the same area. Use rectangle tool to draw object around band.Clean plate with dI and kim wipes, then with ethanol from the glass bottle.
HIS TAGGED DHFR FROM E COLI WESTERN BLOT RESULTS SOFTWARE
Leave software open if anyone is signed up within two hours. Remove plate before shutting down scanner.Transfer blot face down to fluorimager plate. Using forceps, lay blot face down on top of substrate (no bubbles) for about one minute (less if bands become visible).ECF substrate in buffer is stored in 1 mL aliquots at –80C. Place 1 mL/gel of ECF substrate on a transparency.Turn on Fluorimager 30 minutes before use.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.Incubate overnight with 1:10000 dilution of anti-GFP in 2% milk/TBS-Tween (0.1%).(for two blots, use a large tip box and 25 mL fluid for all incubations/washes) Place blot on shaker table for all incubations. Use the lid of a pipette tip box and 15 mL fluid to cover blot. Block membrane for 1 hr with 5% milk/TBS-Tween (0.1 %).For one 8.5 X 5 blot, this works out to 85 mA for about 2 hours. rainbow marker is fully transferred to PVDF membrane). Transfer 2 mA/cm^2 until transfer is complete (i.e.Be sure to preserve orientation of blot and avoid bubbles. Set up transfer bottom to top: 2 pieces blotting paper, gel, PVDF, two pieces blotting paper.Wet 4 pieces of blotting paper in transfer buffer. Cut corner on PVDF and wet with methanol, then soak 5 minutes in H20, followed by 10 minutes in transfer buffer.Cut PVDF and 3mm Whatman blotting paper to correct size.After running gel, cut away stacking gel, cut a corner to mark orientation, and soak in transfer buffer for 45 minutes.Run 3 uL Amersham Rainbow marker #755 as size marker. (20 ng/uL purified GFP aliquots at –80C keep on ice until use) control sup’n) to a volume of 5 uL mixed with 5 uL 2X sample buffer. Run GFPssrA standards (10 ng, 20 ng, 40 ng, 60 ng, 80 ng) in water (or neg. Adjust for higher or lower expression levels. Run ~1 E7 cells per lane (5 uL of lysis sup’n and 5 uL of 2X sample buffer). Spin down and load onto tric-tricine acrylamide gel (see JcBAcrylGel_protocol) immediately. Mix samples and standards with 2X sample buffer (in pcr tubes) and boil 10 minutes (95 C heat block).Boil samples for 10 minutes (use 95 C sand block).See tric-tricine acrylamide gel protocol for pouring vertical acrylamide gels Cell lysates can be frozen at this point (-20 C).This concentration is about right for MC4100-pSB4A3.I7101 adjust as necessary. Final cell concentration will be 2 E6 cells/uL (use OD/cfu curve).Immediately spin the cells 13 K (max on table top centrifuge) for 2 minutes to pellet.Start new culture at OD 0.02 from an exponentially growing culture when it reaches ~0.4 OD.JCB Protocol for plasmid based GFP batch or chemostat culture 9 Lysis Buffer (12.5 mM Tris pH 6.8, 4% SDS.
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