Team:EPF-Lausanne/Protocol/Silver staining

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== Gel making ==
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==Preparation of Gel==
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=== Gel Ingredients (choose percentage according to the size of the protein) ===
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'''Note:''' Please, adapt the final percentage of polyacrilamide according to the size of the protein.
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 +
 
{| class="wikitable" style="text-align: center; color: black;"
{| class="wikitable" style="text-align: center; color: black;"
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|'''Expected protein size'''
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|'''Final polyacrilamide concentration '''
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|-
|4-40 kDA
|4-40 kDA
|20%
|20%
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|}
|}
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For a Gel percentage of 7.5 % the proportions are the following:
{| class="wikitable" style="text-align: center; color: black;"
{| class="wikitable" style="text-align: center; color: black;"
|'''Separating gel'''
|'''Separating gel'''
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|-
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|'''Volume, en mL'''
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|Gel percentage
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|7.5 %
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|-
|-
|30% Polyacrylamide
|30% Polyacrylamide
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|10 mL
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|10
|-
|-
|1.5M Tris (pH 8.8)
|1.5M Tris (pH 8.8)
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|10 mL
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|10
|-
|-
|10% Ammonium persulfate
|10% Ammonium persulfate
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|0.4 mL
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|0.4
|-
|-
|10% SDS
|10% SDS
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|0.4 mL
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|0.4
|-
|-
|TEMED
|TEMED
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|0.038 mL
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|0.038
|-
|-
|H2O
|H2O
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|19.2 mL
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|19.2
|-
|-
|Total volume
|Total volume
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|40 mL
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|40
|-
|-
-
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For a 5% Stacking gel,
{| class="wikitable" style="text-align: center; color: black;"
{| class="wikitable" style="text-align: center; color: black;"
|'''Stacking gel'''
|'''Stacking gel'''
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|-
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|'''Volume, en mL'''
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|Gel percentage
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|5 %
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|-
|-
|30% Polyacrylamide
|30% Polyacrylamide
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|1.36 mL
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|1.36
|-
|-
|1M Tris (pH 6.8)
|1M Tris (pH 6.8)
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|-
|-
|10% Ammonium persulfate
|10% Ammonium persulfate
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|0.08 mL
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|0.08
|-
|-
|10% SDS
|10% SDS
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|0.08 mL
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|0.08
|-
|-
|TEMED
|TEMED
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|0.008 mL
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|0.008
|-
|-
|H2O
|H2O
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|5.44 mL
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|5.44
|-
|-
|Total volume
|Total volume
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[[File:Team-EPF-Lausanne_ssp.jpg|470px]]
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==Gel Running==
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For running and transfering the gel, please refer to [[Team:EPF-Lausanne/Protocol/Western_Blot|Wester Blot protocol]].
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*Loading Plan
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-
 
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*Lane 1: Ladder
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*Lane 2: Empty well
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*Lane 3: 10microL of VP16 only
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*Lane 4: 25microL of VP16 only
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*Lane 5: Nontransfected CHO cell - High concentration (5million)
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*Lane 6: Sample 1 48h of expression
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*Lane 7: Sample 2 48h of expression
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*Lane 8: Sample 1 72h of expression
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*Lane 9: Sample 2 72h of expression
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*Lane 10: Nontransfected CHO cell - Same concentration as samples
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==Silver Staining==
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[[File:Team-EPF-Lausanne_ssp.jpg|650px]]
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[[File:Team-EPF-Lausanne_21.sep.12 ssr.jpg|600px]]
 
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Result: The lane 9 (Lovtap-VP16 transfected CHO cell) and lane 10 (non-transfected CHO cell) have intensity difference around 30~35kDa area.
 

Latest revision as of 00:07, 27 September 2012

Contents

Protocol: Silver Staining


Preparation of Gel

Note: Please, adapt the final percentage of polyacrilamide according to the size of the protein.


Expected protein size Final polyacrilamide concentration
4-40 kDA 20%
12-45 kDA 15%
10-70 kDA 12.5%
15-100 kDA 10%
25-200 kDA 8%

For a Gel percentage of 7.5 % the proportions are the following:

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


Gel Running

For running and transfering the gel, please refer to Wester Blot protocol.

Silver Staining

Team-EPF-Lausanne ssp.jpg




Wiki Links

Important pages

Pages

Retrieved from "http://2012.igem.org/Team:EPF-Lausanne/Protocol/Silver_staining"