Endy:General Western Blot
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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
- 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.)
volume = (average pixel value – background value) * object area
- 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