Team:EPF-Lausanne/Protocol/Western Blot

From 2012.igem.org

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(→III. Antibody tagging)
 
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{{:Team:EPF-Lausanne/Template/ProtocolHeader|NEW Western Blot|{{{1|}}}}}
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<noinclude>{{:Team:EPF-Lausanne/Template/SetTitle|Western Blot}}</noinclude>
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{{:Team:EPF-Lausanne/Template/ProtocolHeader|Western Blot|{{{1|}}}}}
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=== To prepare for Protein samples ===
 
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1. Centrifuge around 5million cells(Volume doesn't matter) for 2,500x, 10 min.
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=== Gel Ingredients (choose percentage according to the size of the protein) ===
 +
{| class="wikitable" style="text-align: center; color: black;"
 +
|4-40 kDA
 +
|20%
 +
|-
 +
|12-45 kDA
 +
|15%
 +
|-
 +
|10-70 kDA
 +
|12.5%
 +
|-
 +
|15-100 kDA
 +
|10%
 +
|-
 +
|25-200 kDA
 +
|8%
 +
|-
 +
|}
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2. Discard the supernatant with Vacuum chamber.
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 +
{| class="wikitable" style="text-align: center; color: black;"
 +
|'''Separating gel'''
 +
|-
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|Gel percentage
 +
|7.5 %
 +
|-
 +
|30% Polyacrylamide
 +
|10 mL
 +
|-
 +
|1.5M Tris (pH 8.8)
 +
|10 mL
 +
|-
 +
|10% Ammonium persulfate
 +
|0.4 mL
 +
|-
 +
|10% SDS
 +
|0.4 mL
 +
|-
 +
|TEMED
 +
|0.038 mL
 +
|-
 +
|H2O
 +
|19.2 mL
 +
|-
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|Total volume
 +
|40 mL
 +
|-
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3. Mix the cell pellet with 1X PBS and centrifuge it for 2,500x, 10 min.
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 +
{| class="wikitable" style="text-align: center; color: black;"
 +
|'''Stacking gel'''
 +
|-
 +
|Gel percentage
 +
|5 %
 +
|-
 +
|30% Polyacrylamide
 +
|1.36 mL
 +
|-
 +
|1M Tris (pH 6.8)
 +
|1 mL
 +
|-
 +
|10% Ammonium persulfate
 +
|0.08 mL
 +
|-
 +
|10% SDS
 +
|0.08 mL
 +
|-
 +
|TEMED
 +
|0.008 mL
 +
|-
 +
|H2O
 +
|5.44 mL
 +
|-
 +
|Total volume
 +
|8 mL
 +
|-
 +
|}
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4. Discard the supernatant with Vacuum chamber.
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=== Preparing Protein Samples ===
-
5. Necessary amount of lysis buffer depends on the pellet size - for 20mg pellet -> 150micro liters of IP lysis buffer.
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1. Centrifuge around 5 million cells (of any volume) at 2,500 rpm for 10 min.
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6. Put the lysated sample in an ice box for 10 min - flip it off every 3 mins.
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2. Discard the supernatant with a vacuum pump.
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7. Add up 3x SDS lysis buffer (Here I put 75micro liters because the IP lysis buffer amount was 150micro liters)
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3. Resuspend the cell pellet with 1x PBS and centrifuge it at 2,500 rpm for 10 min.
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8. Put the sample in 95 degree of heater to denature proteins for 5 mins.
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4. Discard the supernatant with a vacuum pump.
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=== To prepare loading samples ===
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5. Add appropriate amount of lysis buffer depending on the pellet size (for a 20 mg pellet, 150 µl of IP lysis buffer).
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<!--  -->
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-
1. Volume for 7 micro liter of ladder is enough.
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6. Keep the lysed sample on ice for 10 min - flick every 3 minutes.
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2. Make the sample size up to 50 micro liters.
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7. Add 3x SDS lysis buffer (for a 20 mg pellet, 75 µl).
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Ex) 30microliters of cell lysis, 1microliter of VP16 (10microgram per 1microliter concentration), 19 microliters of SDS lysis buffer = 50 microliters of sample is ready.
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8. Incubate the sample for 5 minutes at 95 degrees, to denature proteins.
 +
 
 +
=== Preparing loading samples ===
 +
<!--  -->
 +
 
 +
1. Load the ladder (7 µl is the recommended volume).
 +
 
 +
2. Complete sample volume to 50 µl.
3. Load the samples.
3. Load the samples.
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=== I. SDS Gel electrophoresis ===
=== I. SDS Gel electrophoresis ===
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1. Prepare the separating gel and stacking gel solution without APS and TEMED.
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1. Prepare the separating and stacking gel solutions without APS and TEMED.
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2. If the SDS kit is ready to work, add APS and TEMED into the separating gel solution and pour a little bit of DW on top of the solution.
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2. Add APS and TEMED to the separating gel solution only when the SDS kit is ready to be used, they are time-sensitive. Move the solution inside of the setup. Add some distilled water on top of it.
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3. After 20~30 mins, take out the DW and check the gel is solidified or not.
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3. After 20-30 mins, remove the water and check whether the gel has solidified. Don't move to the next step until it does.
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4. Put TEMED into the stacking gel solution and pour it on the solidified separating gel.
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4. Add TEMED to the stacking gel solution, pour it on top of the solidified separating gel.
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5. Insert a stack carefully and leave it for 20~30 mins.
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5. Insert a stack carefully and leave it for 20-30 mins.
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6. Take out the stack and fill up SDS loading buffer in the kit.
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6. Take the stack out and fill the kit with SDS loading buffer.
7. Load the samples.
7. Load the samples.
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8. Add more loading buffer and set up the machine with 80volts for 1.5 hours.
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8. Add more loading buffer, set the voltage to 80 Volts. Leave for 1.5 hours.
=== II. Membrane transfer ===
=== II. Membrane transfer ===
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1. Prepare a membrane transfer kit.
1. Prepare a membrane transfer kit.
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2. Take out the gel from the SDS kit and put it on the membrane paper.
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2. Take the gel out of the SDS kit and put it on the membrane paper.
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3. From the bottom to top, put in order of 1) Sponge - 2) Blot paper - 3) Membrane - 4) Gel (Now pour some M-transfer buffer to make it fully wet) - 5) Blot paper again - 6) Sponge again.
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3. From bottom to top, assemble the components in the following order: 1) Sponge - 2) Blot paper - 3) Membrane - 4) Gel (Pour some M-transfer buffer on the gel) - 5) Blot paper again - 6) Sponge again.
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4. Close the kit and set up the voltage with 20 for 30mins ~ 1 hour.
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4. Close the sandwich, set the voltage to 20 V. Leave for 30 mins - 1 hour.
 +
 
 +
5. Discard the gel. Leave the membrane in 5% skim milk with 30ml of TBST buffer (blocking buffer, to achieve the 5%, add 1.5 g of skim milk powder to the buffer) for one hour.
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5. Discard the gel and put the membrane in 5% Skim milk with 30ml of TBST buffer(1.5g of skim milk power for 5% here) for an hour.
 
=== III. Antibody tagging ===
=== III. Antibody tagging ===
 +
1. Discard the blocking buffer, leave only 5ml of it. Add primary antibody with a ratio of 1:1000 or 1:2000 (5 µl of antibody in 5 ml of buffer gives 1:1000)
 +
2. Leave the mix overnight at 4 °C.
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1. Leave 5ml of the 5% skim milk buffer and mix it up with 1:2000 or 1:1000 of primary antibody(5micro liters of antibody : 5ml of buffer = 1: 1000)
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3. Wash 3 times with 1x TBST (5 minutes on shaker for every wash).
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+
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2. Overnight it at 4 degree.
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-
 
+
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3. Wash it out with 1X TBST for 5 times for 5 mins each.
+
-
4. With 5% skim milk buffer, use 1:2000 dilution of secondary antibody (we have goat anti rabbit antibody) and leave it in room temperature for 2 hours.
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4. Dilute the secondary antibody (for example, goat anti-rabbit antibody) to 1:2000 in 5% skim milk buffer. Add it. Leave at room temperature for 2 hours.
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5. Wash it out with 1X TBST for 5 times for 5mins each.
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5. Wash 3 times with 1x TBST (5 minutes on shaker for every wash).
-
6. Check the protein level in the dark room.
+
6. Reveal the protein bands in the dark room.
<!-- Don't forget to add this protocol to the protocol list page: -->
<!-- Don't forget to add this protocol to the protocol list page: -->

Latest revision as of 12:56, 22 September 2012

Contents

Protocol: Western Blot

Gel Ingredients (choose percentage according to the size of the protein)

4-40 kDA 20%
12-45 kDA 15%
10-70 kDA 12.5%
15-100 kDA 10%
25-200 kDA 8%


Separating gel
Gel percentage 7.5 %
30% Polyacrylamide 10 mL
1.5M Tris (pH 8.8) 10 mL
10% Ammonium persulfate 0.4 mL
10% SDS 0.4 mL
TEMED 0.038 mL
H2O 19.2 mL
Total volume 40 mL
Stacking gel
Gel percentage 5 %
30% Polyacrylamide 1.36 mL
1M Tris (pH 6.8) 1 mL
10% Ammonium persulfate 0.08 mL
10% SDS 0.08 mL
TEMED 0.008 mL
H2O 5.44 mL
Total volume 8 mL

Preparing Protein Samples

1. Centrifuge around 5 million cells (of any volume) at 2,500 rpm for 10 min.

2. Discard the supernatant with a vacuum pump.

3. Resuspend the cell pellet with 1x PBS and centrifuge it at 2,500 rpm for 10 min.

4. Discard the supernatant with a vacuum pump.

5. Add appropriate amount of lysis buffer depending on the pellet size (for a 20 mg pellet, 150 µl of IP lysis buffer).

6. Keep the lysed sample on ice for 10 min - flick every 3 minutes.

7. Add 3x SDS lysis buffer (for a 20 mg pellet, 75 µl).

8. Incubate the sample for 5 minutes at 95 degrees, to denature proteins.

Preparing loading samples

1. Load the ladder (7 µl is the recommended volume).

2. Complete sample volume to 50 µl.

3. Load the samples.

I. SDS Gel electrophoresis

1. Prepare the separating and stacking gel solutions without APS and TEMED.

2. Add APS and TEMED to the separating gel solution only when the SDS kit is ready to be used, they are time-sensitive. Move the solution inside of the setup. Add some distilled water on top of it.

3. After 20-30 mins, remove the water and check whether the gel has solidified. Don't move to the next step until it does.

4. Add TEMED to the stacking gel solution, pour it on top of the solidified separating gel.

5. Insert a stack carefully and leave it for 20-30 mins.

6. Take the stack out and fill the kit with SDS loading buffer.

7. Load the samples.

8. Add more loading buffer, set the voltage to 80 Volts. Leave for 1.5 hours.

II. Membrane transfer

1. Prepare a membrane transfer kit.

2. Take the gel out of the SDS kit and put it on the membrane paper.

3. From bottom to top, assemble the components in the following order: 1) Sponge - 2) Blot paper - 3) Membrane - 4) Gel (Pour some M-transfer buffer on the gel) - 5) Blot paper again - 6) Sponge again.

4. Close the sandwich, set the voltage to 20 V. Leave for 30 mins - 1 hour.

5. Discard the gel. Leave the membrane in 5% skim milk with 30ml of TBST buffer (blocking buffer, to achieve the 5%, add 1.5 g of skim milk powder to the buffer) for one hour.

III. Antibody tagging

1. Discard the blocking buffer, leave only 5ml of it. Add primary antibody with a ratio of 1:1000 or 1:2000 (5 µl of antibody in 5 ml of buffer gives 1:1000)

2. Leave the mix overnight at 4 °C.

3. Wash 3 times with 1x TBST (5 minutes on shaker for every wash).

4. Dilute the secondary antibody (for example, goat anti-rabbit antibody) to 1:2000 in 5% skim milk buffer. Add it. Leave at room temperature for 2 hours.

5. Wash 3 times with 1x TBST (5 minutes on shaker for every wash).

6. Reveal the protein bands in the dark room.



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