Fluorescence on NF-films - Proteins - post electrophoresis
April 03, 2025
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.
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)
EDCElectrophoresis Development & Consulting, Vor dem Kreuzberg 17, 72070 Tübingen (Germany)