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Electrophoresis
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Dr. Hanspeter Schickle
Wolfgang Gstrein

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Fluorescence on NF-films - Proteins - post electrophoresis

April 28, 2023

The following protocols below describes the use of the fluorophore “Lava Purple” (Serva, Heidelberg) with proteins on non-fluorescence film-supported gels (SDS - IEF)
The more elegant method is the labeling of the proteins before electrophoresis. Cooperation with Dyeagnostics.

Labeling before electrophoresis on normal support ---> T-REX on Normal Support Protein, Pro-Q on Normal Support Pro-Q


	ElphoGel SDS Gradient Kit: 8.5 cm separation lengths

Solutions for LavaPurple:

Solution 1: 15% ethanol, 1% citric acid
Place 850 mL MilliQ or equivalent and 150 mL AR grade or higher ethanol into a 1 L bottle and add 10g citric acid (1 sachet part A) mix until dissolved.
pH should be between 2.1 and 2.4 (no need to check usually)

Solution 2: 100 mM sodium borate pH 10.9
Dissolve 6.2 g boric acid (1 sachet part B) and 3.84g sodium hydroxide (part C) into 1 L MilliQ or similar water.
All reagents should be ACS grade or similar. pH should be between 10.8 and 11.2. (no need to check usually).

Solution 3: 15% ethanol
Dissolve 150 mL AR grade or higher ethanol into 850 mL MilliQ grade or similar water.

Staining solution:
Dissolve 1 mL LavaPurple concentrate into 200 mL Solution 2
NOTE: It is essential that the concentrate has been brought to room temperature and thoroughly mixed prior to being dissolved.. The solution must be made fresh (no more than 30 minutes prior to use).
The concentrate must be dissolved into solution 2 BEFORE being added to the gel, if the concentrate is added to the tray staining artifacts will occur.


	SDS-protocol normal size gel (12 x 26 cm), 0.65 mm thick:

Process	Solution	Time	Tray	Temp
Pre-fixing	Recycled Acidifyer	30 min	300 ml	ambient
Fixing	Solution 1	30 min - overnight	300 ml	ambient
Pre-buffering 1	Recycled Staining solution	15 min	300 ml	ambient
Pre-buffering 2	Solution 2	15 min	300 ml	ambient
Staining	Staining solution	60 - 90 min	200 ml	ambient
Destaining	Solution 3	30 min	300 ml	ambient
Acidifying	Solution 1	30 min	300 ml	ambient

Prefix with the recycled Acidifyer for 30 min, then fix gel in 300 mL Solution 1 for 30 min or overnight.
The gel may be fixed overnight with no ill effects. Removing concentrate from -20°C at this time will ensure the solution is ready for use.
First Pre-buffering step could be done with the used and recycled Staining solution.
Prepare 1 × staining solution (200 mL) and stain gel for 1-1.5 hr.
It is not necessary to protect the gel from light. DO NOT stain longer than 3 hrs as signal will decrease with time.
(Recycling of the used staining solution is recommended for the first pre-buffering step)
De-stain gel in 300 mL Solution 3 for 30 minutes.
Acidify gel in 300 mL Solution 1 for 30 minutes.
Note: If background from samples is particularly high, the times for each step should be increased to 1.5 hr for steps 1 and 2 and 45 minutes for steps 3 and 4. (Recycling of the used Acidifyer is recommended for the Pre-Fixing step)

SDS-protocol large size gel (20 x 26 cm), 1 mm thick:

Process	Solution	Time	Tray	Temp
Pre-fixing	Recycled Acidifyer	45 min	1000 ml	ambient
Fixing	Solution 1	45 min - overnight	1000 ml	ambient
Pre-buffering 1	Recycled Staining solution	20 min	1000 ml	ambient
Pre-buffering 2	Solution 2	20 min	1000 ml	ambient
Staining	Staining solution	1 h 30 min	600 ml	ambient
Destaining	Solution 3	45 min	400 ml	ambient
Acidifying	Solution 1	45 min	400 ml	ambient

Prefix with the recycled Acidifyer for 45 min, then fix gel in 1000 mL Solution 1 for 45 min or overnight.
The gel may be fixed overnight with no ill effects. Removing concentrate from -20°C at this time will ensure the solution is ready for use.
First Pre-buffering step could be done with the used and recycled Staining solution.
Prepare 1 × staining solution (600 mL) and stain gel for 1.5 hr.
It is not necessary to protect the gel from light. DO NOT stain longer than 3 hrs as signal will decrease with time.
(Recycling of the used staining solution is recommended for the first pre-buffering step)
De-stain gel in 400 mL Solution 3 for 45 minutes.
Acidify gel in 400 mL Solution 1 for 45 minutes.
(Recycling of the used Acidifyer is recommended for the Pre-Fixing step)


	IEF Protocol Normal size gel (12 x 26 cm), 0.65 mm thick:

Process	Solution	Time	Tray	Temp
Fixing	20% TCA	45 min	200 ml	ambient
Pre-Washing	Recycled Staining solution	10 min	200 ml	ambient
Washing	Solution 2	10 min	200 ml	ambient
Staining	Staining solution	60 min	300 ml	ambient
Destaining	Solution 3	30 min	300 ml	ambient
Acidifying	Solution 1	30 min	300 ml	ambient

Fix gel in 200 mL 20% TCA for 45 minutes.
Removing concentrate from -20 degrees C at this time will ensure the solution is ready for use.
Prewash the gel in recycled Staining solution, then wash gel in 200 mL Solution 2 for 10 minutes.
Prepare 1 × staining solution (200 mL) and stain gel for 1 hr.
It is not necessary to protect the gel from light. DO NOT stain longer than 3 hrs as signal will decrease with time.
(Recycling of the used staining solution is recommended for the pre-washing step)
De-stain gel in 300 mL Solution 3 for 30 minutes.
Acidify gel in 300 mL Solution 1 for 30 minutes.
Note: If background from samples is particularly high the time should be increased to 1.5 hr for step 2 and 45 minutes for steps 3 and 4.


	Scanning: using Typhoon 9200 (532 nm laser, 540 PMT, 610BP30 filter, 100 µm resolution, normal sensitivity)

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