ESO wikiadmin at 12:39, 27 February 2025

2025-02-27T12:39:16Z

← Older revision		Revision as of 14:39, 27 February 2025
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	!Materials		!Materials
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	Luciferin aliquots (-20°C freezer)		Luciferin aliquots (-20°C freezer)
	Multipipette and plastic box for solution (cell culture room)		Multipipette and plastic box for solution (cell culture room)
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	\|Assay buffer (100ml):		\|Assay buffer (100ml):
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	20 mM MgSO4 [1 M] → 2 ml		20 mM MgSO4 [1 M] → 2 ml
	adjust pH with phosphoric acid again or KOH/NaOH to definitely get pH 7.8 shortly before use: add 10 µl = 1 mM DTT [1 M] and 100 µl = 2 mM ATP [200 mM] per 10 ml		adjust pH with phosphoric acid again or KOH/NaOH to definitely get pH 7.8 shortly before use: add 10 µl = 1 mM DTT [1 M] and 100 µl = 2 mM ATP [200 mM] per 10 ml
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	\|Lysis buffer (100ml):		\|Lysis buffer (100ml):
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	1 % Triton X-100 → 1 ml 10% glycerol → 10 ml		1 % Triton X-100 → 1 ml 10% glycerol → 10 ml
	shortly before use: add 2 µl/ml = 2 mM DTT [1 M]		shortly before use: add 2 µl/ml = 2 mM DTT [1 M]
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	\|D-Luciferin (110 ml):		\|D-Luciferin (110 ml):
	\|Dissolve 0.17g DTT in 110 ml ddH2O, take 1 ml of this solution and add to 25 mg D-Luciferin, add 20 µl of 10N NaOH for dissolving Luciferin (careful: too much causes oxidation! → green solution), dilute premixture into remaining 109 ml DTT solution, aliquot and freeze at -20°C!		\|Dissolve 0.17g DTT in 110 ml ddH2O, take 1 ml of this solution and add to 25 mg D-Luciferin, add 20 µl of 10N NaOH for dissolving Luciferin (careful: too much causes oxidation! → green solution), dilute premixture into remaining 109 ml DTT solution, aliquot and freeze at -20°C!
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ESO wikiadmin: Created page with "{| class="wikitable" |+ !Materials ! ! ! |- | |Ice bath for buffers, Luciferin and lysates PBS rubber policeman (cold room) and Eppis for lysates, Eppi centrifuge at RT Lysis buffer (1M DTT → 2 µl per ml) (fridge 4°C) Assay buffer (1M DTT → 10 µl per 10 ml; 200 mM ATP → 100ul per 10 ml) (fridge 4°C) Luciferin aliquots (-20°C freezer) Multipipette and plastic box for solution (cell culture room) | | |- |Assay buffer (100ml): |25 mM Tris pH 7.8 [1 M] → 2.5 ml..."

2025-02-27T12:38:59Z

Created page with "{| class="wikitable" |+ !Materials ! ! ! |- | |Ice bath for buffers, Luciferin and lysates PBS rubber policeman (cold room) and Eppis for lysates, Eppi centrifuge at RT Lysis buffer (1M DTT → 2 µl per ml) (fridge 4°C) Assay buffer (1M DTT → 10 µl per 10 ml; 200 mM ATP → 100ul per 10 ml) (fridge 4°C) Luciferin aliquots (-20°C freezer) Multipipette and plastic box for solution (cell culture room) | | |- |Assay buffer (100ml): |25 mM Tris pH 7.8 [1 M] → 2.5 ml..."

New page

{| class="wikitable"
|+
!Materials
!
!
!
|-
|
|Ice bath for buffers, Luciferin and lysates PBS
rubber policeman (cold room) and Eppis for lysates, Eppi centrifuge at RT Lysis buffer (1M DTT → 2 µl per ml) (fridge 4°C)
Assay buffer (1M DTT → 10 µl per 10 ml; 200 mM ATP → 100ul per 10 ml) (fridge 4°C)
Luciferin aliquots (-20°C freezer)
Multipipette and plastic box for solution (cell culture room)
|
|
|-
|Assay buffer (100ml):
|25 mM Tris pH 7.8 [1 M] → 2.5 ml
4 mM EGTA [100 mM] → 4 ml
20 mM MgSO4 [1 M] → 2 ml
adjust pH with phosphoric acid again or KOH/NaOH to definitely get pH 7.8 shortly before use: add 10 µl = 1 mM DTT [1 M] and 100 µl = 2 mM ATP [200 mM] per 10 ml
|
|
|-
|Lysis buffer (100ml):
|25 mM Tris pH 7.8 [1 M] → 2.5 ml
4 mM EGTA [100 mM] → 4 ml
1 % Triton X-100 → 1 ml 10% glycerol → 10 ml
shortly before use: add 2 µl/ml = 2 mM DTT [1 M]
|
|
|-
|D-Luciferin (110 ml):
|Dissolve 0.17g DTT in 110 ml ddH2O, take 1 ml of this solution and add to 25 mg D-Luciferin, add 20 µl of 10N NaOH for dissolving Luciferin (careful: too much causes oxidation! → green solution), dilute premixture into remaining 109 ml DTT solution, aliquot and freeze at -20°C!
|
|
|}

=== Procedure ===
----

* Wash transfected cells (HEK 293) with PBS, add lysis buffer (1.3 ml per 10 cm dish), incubate at 4°C, 15 min
* scrape cells with rubber policeman, transfer to Eppi tubes, homogenization of DNA with syringe
* Centrifugation at 13 500 rpm, 5 min, RT
* Prime and install Luciferin solution at dispenser of Varioscan plate reader; submit 10-15 µl of supernatant in each well of white 96-well reader plate and measure fluorescence (mKate 588/633 nm). Add Assay buffer with multipipette (for 15 µl lysate 215 µl)
* After dispensing D-Luciferin solution (25 µl for 15 µl lysate) measure luminescence with different parameters or perform a kinetic curve (design your own protocol for your purpose)
* Data Analysis L/mKate (normalization)

Luciferase Reporter Assay - Revision history

ESO wikiadmin at 12:39, 27 February 2025