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Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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Fluorescence on NF-films - Proteins - post electrophoresis

Mai 30, 2018

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.

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

2D-DryGel Kit 2x7/11: 2x11 cm IPG. Run on flatbed basic

2D-DryGel Kit 2x7/11 post-electrophoresis staining

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)