Western blot protocol (Conlon lab)

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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.