Endy:E. coli Western Blot: Difference between revisions
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modification of Chris Farrell’s protocol | modification of Chris Farrell’s protocol | ||
==Sample preparation== | ==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== | ==Gel Preparation== | ||
see tric-tricine acrylamide gel protocol for pouring vertical acrylamide gels | 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 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. | *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) | (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). | *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] | [1 E7 cell/lane is good for pSB4A3.I7101. Adjust for higher or lower expression levels] | ||
==Transfer and Incubations== | ==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== | ==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== | ==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 | 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== | ==2X Tricine Sample Buffer== | ||
2 mL 4X Tris-Cl/SDS, pH 8.8 | *2 mL 4X Tris-Cl/SDS, pH 8.8 | ||
6 mL 40% glycerol (24% final) | *6 mL 40% glycerol (24% final) | ||
0.8 g SDS (8% final) | *0.8 g SDS (8% final) | ||
0.31 g DTT (0.2 M final) | *0.31 g DTT (0.2 M final) | ||
2 mg Coomassie blue G-250 (0.02% final) (used C. Blue G) | *2 mg Coomassie blue G-250 (0.02% final) (used C. Blue G) | ||
to 10 mL with MilliQ H2O and mix | *to 10 mL with MilliQ H2O and mix | ||
aliquot 500 uL/tube and store at –20 C | *aliquot 500 uL/tube and store at –20 C | ||
==4X Tris-Cl/SDS pH 8.8== | ==4X Tris-Cl/SDS pH 8.8== | ||
91 g Tris | *91 g Tris | ||
dissolve in 300 mL H2O | *dissolve in 300 mL H2O | ||
pH to 8.8 with 1N HCl (about 120 mL) | *pH to 8.8 with 1N HCl (about 120 mL) | ||
to 500 mL with H2O | *to 500 mL with H2O | ||
filter 0.45 um | *filter 0.45 um | ||
add 2g SDS and store 4 C | *add 2g SDS and store 4 C | ||
==Lysis Buffer (12.5 mM Tris pH 6.8, 4% SDS== | ==Lysis Buffer (12.5 mM Tris pH 6.8, 4% SDS== | ||
1.25 mL 1M Tris (pH 8) | *1.25 mL 1M Tris (pH 8) | ||
to 80 mL with st. H2O | *to 80 mL with st. H2O | ||
pH if necessary | *pH if necessary | ||
to 100 mL with st. H2O | *to 100 mL with st. H2O | ||
add 4g SDS | *add 4g SDS | ||
==TBS-Tween (0.1%)== | ==TBS-Tween (0.1%)== | ||
100 mL 10X TBS | *100 mL 10X TBS | ||
900 mL H2O | *900 mL H2O | ||
1 mL Tween (Polyoxyethylene sorbitan monolaurate) | *1 mL Tween (Polyoxyethylene sorbitan monolaurate) | ||
10X TBS (500 mM Tris, 1.5 M NaCl) | *10X TBS (500 mM Tris, 1.5 M NaCl) | ||
150 mL 5M NaCl | **150 mL 5M NaCl | ||
250 mL 1M Tris, pH 7.5 | **250 mL 1M Tris, pH 7.5 | ||
to 500 mL with H2O | **to 500 mL with H2O | ||
==1M Tris-Cl, pH 8 (or 7.5)== | ==1M Tris-Cl, pH 8 (or 7.5)== | ||
121 g tris base | *121 g tris base | ||
700 mL MilliQ H2O | *700 mL MilliQ H2O | ||
to pH 8 with 6N HCl (about 100 mL) | *to pH 8 with 6N HCl (about 100 mL) | ||
to 1 L with MilliQ H2O | *to 1 L with MilliQ H2O | ||
filter 0.45 um or autoclave (fluid, 20 psi, 250 F, 20 min) | *filter 0.45 um or autoclave (fluid, 20 psi, 250 F, 20 min) | ||
==Transfer Buffer (“Towbin Buffer”)== | ==Transfer Buffer (“Towbin Buffer”)== | ||
3 g Tris | *3 g Tris | ||
14.4 g glycine | *14.4 g glycine | ||
800 mL dI | *800 mL dI | ||
200 mL methanol | *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) | 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) | ECF Western Blotting kit is Amersham RPN5783 (rabbit) | ||
To connect to Bionet from fluorimager: \\18.79.1.147\endy DNA-NET\username | To connect to Bionet from fluorimager: \\18.79.1.147\endy DNA-NET\username |
Revision as of 19:43, 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