SDS-PAGE gels (Conlon lab) and Western blot protocol (Conlon lab): Difference between pages

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imported>Virgilio
(Created page with "Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot] link to protocol document [http://www.unc.edu/~fconlon/Protocols/SDS_pag...")
 
imported>Virgilio
(Created page with "Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot] link to protocol document [http://www.unc.edu/~fconlon/Protocols/Wester...")
 
Line 1: Line 1:
Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot]
Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot]


link to protocol document [http://www.unc.edu/~fconlon/Protocols/SDS_page.docx]


link to protocol document [http://www.unc.edu/~fconlon/Protocols/Western%20and%20lysis.doc]


CASTING A SDS-PAGE (DENATURING) GEL


Western Protocol


'''IMPORTANT Clean glass spacers and plates prior to and IMMEDIATELY after use! Failure to do so will result in uneven gel preparation, cracking of glass plates, and leaking of gel before polymerization. '''
(updated 3-17-2011)


1. Clean sufficient amount of glass spacers and plates for your experiment (If you are running two gels, clean two spacers and two plates). Clean first with 95% ethanol and wipe clean with kimwipe, then clean with distilled water, wipe clean. Allow to air-dry sufficiently on paper towel until ready to cast gel.


2. Prepare stacking and resolving gel solutions, minus the APS and TEMED, which polymerize the gel. For protein gel applications, we use Bio-Rad bisacrylamide #161-0148, pore size 37.5:1.  BE SURE TO CHECK % of stock solution before using.  The % resolving  gel you use should be determined by the sizes of proteins you are attempting to resolve. 7.5% is better for viewing larger proteins, 120kDa and up.  15% is better for viewing smaller proteins ( i.e. <50 kDa). The stacking gel is fixed at 5% acrylamide. This allows all the proteins in your sample to run evenly into the resolving gel.
FOR EMBRYOS:


:CAUTION: Bisacrylamide in liquid form is really bad for you. Use extreme caution to make sure you don’t spill it, especially on yourself. Once polymerized to solid state, it is no longer a danger.
Lysis Buffer (10 ml)


Recipe for gels (adjust water and acrylamide for 40% stock):
Resolving  Gels (15 ml) 
{|border="1" cellpadding="5"
{|border="1" cellpadding="5"
|-
|-
|
! scope="row" style="background:#efefef;" |Lysis Buffer (10 ml)
! scope="col" style="background:#efefef;" | 7.50%
|500ul 1M Tris pH 7.6
! scope="col" style="background:#efefef;" | 10%
! scope="col" style="background:#efefef;" | 12%
! scope="col" style="background:#efefef;" | 15%
|-
! scope="row" style="background:#efefef;" | 1.5M Tris pH 8.8
|5 ml
|5 ml
|5 ml
|5 ml
|-
|-
! scope="row" style="background:#efefef;" | 30% acrylamide
! scope="row" style="background:#efefef;" |150mM NaCl
|3.75 ml
|200ul 0.5M EDTA
|4 ml
|6 ml
|6 ml
|-
|-
! scope="row" style="background:#efefef;" | water
! scope="row" style="background:#efefef;" |10mM EDTA
|6.15 ml
|200ul 0.5M EDTA
|5.9 ml
|3.9 ml
|2.4 ml
|-
|-
! scope="row" style="background:#efefef;" | 20% SDS
! scope="row" style="background:#efefef;" |1% TritonX-100
|75 ul
|1 ml 10% TritonX-100
|75 ul
|75 ul
|75 ul
|-
|-
! scope="row" style="background:#efefef;" | 10% APS
! scope="row" style="background:#efefef;" |Protease inhibitors
|75 ul
|1 Roche complete mini tablet (4°)
|75 ul
|75 ul
|75 ul
|-
! scope="row" style="background:#efefef;" | TEMED
|25 ul
|25 ul
|25 ul
|25 ul
|}
|}




Stacking Gels (5 ml)


{|border="1" cellpadding="5"
Lysis and SDS PAGE
|-
 
! scope="row" style="background:#efefef;" |0.5M Tris pH 6.8
1. Turn on Lieb lab water bath biorupter about 30min before lysis.
|.62 ml
 
|-
2. Lyse embryos on ice in 100ul of cold lysis buffer by pipetting up and down.
! scope="row" style="background:#efefef;" |30% acrylamide
 
|.833 ml
3. Sonicate samples for 10min on High setting: 30sec on/ 30sec off.
|-
 
! scope="row" style="background:#efefef;" |0.5M Tris pH 6.8
4. Spin samples at 4° at 14,000 rpm for 5min.
|.62 ml
 
|-
5. Move supernatant to new tube. 
! scope="row" style="background:#efefef;" |water
 
|3.817 ml
6. Normalize loading volumes by performing a Coomassie protein assay for all samples.
|-
 
! scope="row" style="background:#efefef;" |20% SDS
7. For maximum loading volume combine 17.75ul protein (17.75ul of sample with lowest concentration, then normalize other samples to this using water to make up the difference) with 6.25ul 4X Loading Dye and 1ul BME. 
|25 ul
 
|-
8. Boil samples for 6-10 minutes.
! scope="row" style="background:#efefef;" |10% APS
 
|50 ul
9. Spin samples at 14,000 rpm, RT for 3 min.
|-
 
! scope="row" style="background:#efefef;" |TEMED
10. Load 25ul of sample onto gel for western analysis.
|5 ul
 
|}
 
FOR TISSUE CULTURE:
 
:RIPA Buffer (100 mL – can store at 4°)
 
:Add 790mg Tris base to 75mL diH20. (50mM Tris-HCl, pH 7.4)
 
:Add 900mg NaCl and stir until all solids are dissolved. (150mM NaCl)
 
:Using HCl, adjust pH to 7.4
 
:Add 10mL 10% NP-40 (1% NP-40)
 
:Add 2.5mL 10% Na-deoxycholate and stir until solution is clear (0.25% Na-deoxych.)
 
:Add 1mL 100mL EDTA.  (1mM EDTA)
 
:Bring to 100mL with diH20.
 
 
At time of use, add protease inhibitors
 
Lysis and SDS PAGE
 
1. Aspirate media from cells. Wash cells with ice cold PBS 1X.
 
2. Add 200uL RIPA/well of 6-well dish (just enough RIPA to barely cover cells)
 
3. Rock plate at 4° for 5min.
 
4. Rinse cells off plate using RIPA buffer. Alternately, scrape cells from well in PBS, then spin at 1500 rpm for 10min at 4°, aspirate PBS from pellet, and resuspend pellet in desired amount of RIPA.
 
5. Transfer lysate to eppendorf. Sonicate samples for 5min (30 sec on/30 sec off) on High in Lieb lab biorupter.
 
6. Spin samples at 14,000 rpm for 5min at 4°.
 
7. Move supernatant to new tube.
 
8. Normalize loading volumes by performing a Coomassie protein assay for all samples.
 
9. Combine desired amount of protein (minimum of 10ug) up to 17.75ul (normalize all samples so that each samples contains the same amount of total protein) with 6.25ul 4X Loading Dye and 1ul BME.
 
10. Boil samples 6-10 minutes.
 
11. Spin samples at 14,000rpm for 3min at RT.
 
12. Load 25ul onto gel.
 
 
Transfer
 
1.  Build “gel sandwich” in transfer buffer using two pieces of Whatman paper, a nitrocellulose membrane, and two sponges.  I find it easiest to build using this orientation:  Gray side-sponge-Whatman paper-gel-nitrocellulose-Whatman paper-sponge-clear side.  Gray side faces black electrode! Don’t forget to orient the gel on the membrane so that the lanes do not transfer backwards. 
 
 
Transfer Buffer
 
:15g glycine
 
:3.5g Tris base


:Bring to 800ml with diH20


These recipes can make up to 4 gels – making this much for 2-3 gels is not a bad idea. This way you ensure you have enough solution in case you have leakage issues.
:Add 200ml MeOH


3. Assemble gel spacers and holders in mini-gel casting apparatus.


4. Add 10% APS and TEMED to Resolving Gel Solution and mix well.  Using P1000, add  1 ml solution at a time in between assembled spacer and plate.  Leave about ¾ inch space from top of the plate (or just fill to top green horizontal bar on gel holder).
2.  Transfer gel ON at 30V or for 1 hr at 400 mA at 4°.   


5. Immediately add 100% EtOH to overlay the resolving gel. This will remove any bubbles in the gel. EtOH does not mix with the acrylamide, so as the acrylamide polymerizes, the EtOH will remain atop the gel. Allow to polymerize at least 10 minutes.
3. Transfer membrane to TBST for storage or to blocking solution.


6. Rinse off EtOH will copius amounts of distilled water. Ensure there is no water left on the resolving gel.


7. Add 10% APS and TEMED to Stacking Gel Solution and mix well.  Using P1000, add  1 ml solution at a time until gel is completely full. AVOID BUBBLES!  Add appropriate gel combs to stacking gel immediately. Again, wait at least 10 minutes for gel to polymerize.
Immunoblotting


8. Assemble gel in electrophoresis chamber. Fill inner chamber with running buffer. Remove gel combs, and fill inner chamber again. There should be no need to clear out wells, there should be no acrylamide or bubbles in them. Load gel and run. Recommended: Run 70V for about 20-30 min until sample has run through the stacking gel. At that point, voltage can be increased to 150V to 200V if needed. Better resolution will occur at lower voltages!
1. Block membrane in 5% dry milk in TBST for at least 1 hour at RT or ON at 4°


9. Once gel has finished running, gently pop glass plate off the spacer using the PLASTIC green spatula. Gently remove gel and transfer or Coomassie stain as normal.
2. Wash membrane 3X in TBST, 5-15min/wash


10. Make sure to clean glass plates well after use! A common practice is to soak them in HOT water while tending to immediate needs, such as transfer or staining. Do not leave them for long after you have run the gel, as residual acrylamide on plates will affect casting of subsequent gels. Be sure to clean off with hot water and then DI water to remove any salts. Place in drying rack.
3. Apply primary antibody diluted in Blocking Solution ON at 4° or for 2hrs at RT.


Notes:
4. Wash membrane 3X in TBST, 5-15min/wash


10% APS (Ammonium persulfate) should be made fresh and stored at 40C for up to ~2 weeks. Aliquots can be stored at -200C indefinitely.
5. Apply secondary antibody diluted in 5% milk in TBST for 1h at RT.


TEMED smells bad (or so Erin says). Open and close quickly. Close tightly.
6. Wash membrane 4X in TBST, 5-15min/wash.


Gels can be cast and left overnight at 4 degrees. To do so, wrap tightly with wet paper towels and again with plastic wrap to keep gel from drying. Keep in mind, this should be done minimally as the number of plates are limited.
7. Develop with ECL.

Latest revision as of 12:54, 22 November 2011

Protocol submitted by VGP from Conlon lab protocols [1]


link to protocol document [2]


Western Protocol

(updated 3-17-2011)


FOR EMBRYOS:

Lysis Buffer (10 ml)

Lysis Buffer (10 ml) 500ul 1M Tris pH 7.6
150mM NaCl 200ul 0.5M EDTA
10mM EDTA 200ul 0.5M EDTA
1% TritonX-100 1 ml 10% TritonX-100
Protease inhibitors 1 Roche complete mini tablet (4°)


Lysis and SDS PAGE

1. Turn on Lieb lab water bath biorupter about 30min before lysis.

2. Lyse embryos on ice in 100ul of cold lysis buffer by pipetting up and down.

3. Sonicate samples for 10min on High setting: 30sec on/ 30sec off.

4. Spin samples at 4° at 14,000 rpm for 5min.

5. Move supernatant to new tube.

6. Normalize loading volumes by performing a Coomassie protein assay for all samples.

7. For maximum loading volume combine 17.75ul protein (17.75ul of sample with lowest concentration, then normalize other samples to this using water to make up the difference) with 6.25ul 4X Loading Dye and 1ul BME.

8. Boil samples for 6-10 minutes.

9. Spin samples at 14,000 rpm, RT for 3 min.

10. Load 25ul of sample onto gel for western analysis.


FOR TISSUE CULTURE:

RIPA Buffer (100 mL – can store at 4°)
Add 790mg Tris base to 75mL diH20. (50mM Tris-HCl, pH 7.4)
Add 900mg NaCl and stir until all solids are dissolved. (150mM NaCl)
Using HCl, adjust pH to 7.4
Add 10mL 10% NP-40 (1% NP-40)
Add 2.5mL 10% Na-deoxycholate and stir until solution is clear (0.25% Na-deoxych.)
Add 1mL 100mL EDTA. (1mM EDTA)
Bring to 100mL with diH20.


At time of use, add protease inhibitors

Lysis and SDS PAGE

1. Aspirate media from cells. Wash cells with ice cold PBS 1X.

2. Add 200uL RIPA/well of 6-well dish (just enough RIPA to barely cover cells)

3. Rock plate at 4° for 5min.

4. Rinse cells off plate using RIPA buffer. Alternately, scrape cells from well in PBS, then spin at 1500 rpm for 10min at 4°, aspirate PBS from pellet, and resuspend pellet in desired amount of RIPA.

5. Transfer lysate to eppendorf. Sonicate samples for 5min (30 sec on/30 sec off) on High in Lieb lab biorupter.

6. Spin samples at 14,000 rpm for 5min at 4°.

7. Move supernatant to new tube.

8. Normalize loading volumes by performing a Coomassie protein assay for all samples.

9. Combine desired amount of protein (minimum of 10ug) up to 17.75ul (normalize all samples so that each samples contains the same amount of total protein) with 6.25ul 4X Loading Dye and 1ul BME.

10. Boil samples 6-10 minutes.

11. Spin samples at 14,000rpm for 3min at RT.

12. Load 25ul onto gel.


Transfer

1. Build “gel sandwich” in transfer buffer using two pieces of Whatman paper, a nitrocellulose membrane, and two sponges. I find it easiest to build using this orientation: Gray side-sponge-Whatman paper-gel-nitrocellulose-Whatman paper-sponge-clear side. Gray side faces black electrode! Don’t forget to orient the gel on the membrane so that the lanes do not transfer backwards.


Transfer Buffer

15g glycine
3.5g Tris base
Bring to 800ml with diH20
Add 200ml MeOH


2. Transfer gel ON at 30V or for 1 hr at 400 mA at 4°.

3. Transfer membrane to TBST for storage or to blocking solution.


Immunoblotting

1. Block membrane in 5% dry milk in TBST for at least 1 hour at RT or ON at 4°

2. Wash membrane 3X in TBST, 5-15min/wash

3. Apply primary antibody diluted in Blocking Solution ON at 4° or for 2hrs at RT.

4. Wash membrane 3X in TBST, 5-15min/wash

5. Apply secondary antibody diluted in 5% milk in TBST for 1h at RT.

6. Wash membrane 4X in TBST, 5-15min/wash.

7. Develop with ECL.

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