Riboflavin photo-polymerizes acrylamide for gel electrophoresis

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Matthias Bock, Irina Passow, Harald Saumweber

Ideas, experiments, author: Matthias Bock

Comparison gels: Irina Passow

Carried out in the Cytogenetics group of Prof. Harald Saumweber at the Humboldt-Universität zu Berlin

July 24 - 31, 2012

Inhaltsverzeichnis

Introduction

Sodiumdodecylsulfate assisted polyacrylamide gel electrophoresis (SDS-PAGE) is one of the "standard methods" of molecular biology and is used to separate proteins. The used polymers (gels) are prepared prior to electrophoresis experiments by polymerization of buffered, aqueous solutions of acrylamide and bisacrylamide. Commonly, ammoniumperoxodisulfat (APS) and tetramethylethylenediamine (TEMED or TMEDA) are used to initiate respectively catalyze this polymerization reaction in laboratories. Both substances have repeatedly been reported to be toxic or at least harmful, are thus not available to a public audience without restrictions and require special storage (protection/conservation) as well as special handling. Furthermore they are derived chemically, which makes them comparably costly and, taken together with their toxicity, rather eco-unfriendly. Also, although APS is a potent radical polymerization initiator, it's activity is expected to diminish over time, since the peroxide spontaneously becomes inactive by setting free oxygen, especially upon solution contamination (with dust etc). an effect, that can affect quality and reproduceability of experiments. The quality of both APS and TEMED can in practice only be tested by applying them.

On the other hand, in many studies riboflavin has been reported to generate radicals in aqueous solution upon irradiation with blue to ultraviolet light (UV). In fact, riboflavin is widely used, especially in medicine, e.g. to harden dentin teeth-coating polymer with UV/blue light. Riboflavin is a biochemical dye with yellow to orange color and green fluorescence. It's quality and purity are visible to the eye and can be quantified by photometry. Riboflavin is not harmful, but even essential as vitamin B2. It does not require special storage or handling. The question remained, whether it is possible to obtain a polyacrylamide gel, that is suitable for protein electrophoresis, using riboflavin instead of APS/TEMED.

Materials & Methods

Acrylamide / Bisacrylamide

A pre-mixed aqueous solution of acrylamide:bisacrylamide = 37,5:1 from Carl Roth was used ("Rotiphorese Gel 30"). The solution contains 30% acrylamide and 0,8% bisacrylamide.

Riboflavin

Preliminary polymerization experiments were carried out using food-additive grade Riboflavin (E101) obtained from funfood4you. According to the supplier, the sample contained 25 mass-% riboflavin and 75 mass-% dextrose. Dextrose contamination however seems not to disturb the polymerization.

Comparative studies were carried out using pharmaceutical grade Riboflavin (>99% purity) from Fagron, obtainable without restriction from local pharmacies.

The yellow to orange powder has a good water solubility in the applied amounts, but is not soluble in oil. The required amounts are very low though (few mg).

Riboflavin is a potent dye: To have a rough estimation, we observed approx. OD 0,5 in a solution of 140 mg pure riboflavin in 10 l water, and OD 0,8 in a solution of 3 mg in 50 ml.

In solution, upon irradiation with blue to ultraviolet light (as well as below ordinary white light neon tubes), riboflavin shows lightgreen fluorescence at an intensity, that is visible to the eye. Riboflavin fluorescence seems to reduce (self-quenching?) at high optical densities.

Ultraviolet light source

Polymerization was initiated by irradiation with ultraviolet light (UV).

In the first experiments UV was provided by 10 light emitting diodes (LEDs) with a emission maximum between 340 to 365 nanometers (nm), driven at a constant voltage of 3,2 volts each. Power consumption was neglibly low. Radiation is not harmful in the applied dosages.

Later experiments were carried out on a Faust NU-72 KL 366 nm etidium bromide gel illuminator. Radiation is intense (36 Watt: 6 tubes, 6 Watt each) and causes sunburn.

Results

Riboflavin initiates acrylamide polymerization

A "big" 10% acrylamide/bis gel was prepared (25ml), a solution of 0,1% food-additive grade riboflavin/dextrose was added, the mixture was pipetted into the PAGE apparatus and coated with isopropanol to exclude oxygen.

The gel was homogenously irratiated by UV LEDs through the PAGE apparatus glas (normal glas, not quartz) for a couple of seconds, then left on the table in a lighted room (neon bulbs, negligle sunlight). Checked after 15 minutes, meniscus was stiff. Continuously UV-irradiated for 5 minutes to test for further polymerization. No additional polymerization was visible, but photobleaching to transparency was observed in the UV focal spot, while the surrounding gel remained yellow. This photobleaching appeared to recover to homogenous yellow color within a larger timescale.

Another "small" acrylamide/bisacrylamide gel was prepared (10ml), using a 0,01% riboflavin/dextrose solution.

Again, the gel was UV-irratiated for a couple of seconds, then left on the table in a lighted room. Checked after 15 minutes, no polymerization had occured. Several flashes from a external camera flash were applied, to test whether intense, short light flashes are suffiencient to initiate polymerization. Checked after 15 minutes, no polymerization had occured. Continous UV-irradiation was applied. Checked after 5 minutes, meniscus was stiff. When disassembling the apparatus, the gel turned out more sticky than before, polymerization seemed not to be as complete. The apparatus was re-assembled and UV-irradiated for another 10 minutes. The gel was now stiff.

We conclude, that a "sufficient" amount of riboflavin is required to initiate polymerization.

However, it appears, ODs > 0,8 inhibit polymerization, rather than enhancing it.

Protocol for a riboflavin SDS-PAGE gel

A few mg of riboflavin were dissolved in water to an OD of 0,8 at 340nm. Pharmaceutical grade riboflavin was used to avoid interference in protein run or subsequent gel staining.

  • separation gel mixture, recipe for 10ml:
10% gel for separation APS/TEMED gel Riboflavin gel
acrylamide/bisacrylamide 37,5:1 3,330 ml 3,330 ml
2M Tris buffer pH 8,8 1,880 ml 1,880 ml
SDS 20% 0,050 ml 0,050 ml
APS 10% 0,050 ml -
TEMED 0,003 ml -
Riboflavin OD 0,8 @ 340 nm - 1,000 ml
H2O 4,690 ml 3,740 ml
  • although the resulting mixture is visibly yellowish, the absorption when pipetted into a 1 mm flat chamber is so low, that it is hard to distinguish riboflavin and APS/TEMED gel with the eye
  • pipette the mixture into the chamber
  • coat to approx. 1 cm with isopropanol to exclude oxygen
  • UV-irradiate for 15 min
  • tilt to check if meniscus is solid
  • if not, irradiate another 15 minutes
  • if yes, mix a stacking gel

Stacking gel polymerization failed

  • stacking gel mixture, recipe for 3ml:
5% top gel APS/TEMED gel Riboflavin gel
acrylamide/bisacrylamide 37,5:1 0,500 ml 0,500 ml
1M Tris buffer pH 6,8 0,375 ml 0,375 ml
SDS 20% 0,015 ml 0,015 ml
APS 10% 0,030 ml -
TEMED 0,003 ml -
Riboflavin OD 0,8 @ 340 nm - 0,100 ml
H2O 2,077 ml 2,010 ml
  • discard isopropanol
  • pipette accumulation gel mixture on top of the separation gel
  • insert sample lane comb, such that the chamber is closed and has no direct contact to air (oxygen)
  • UV-irradiate for 15 min
  • if riboflavin concentration is too low, solution does not polymerize at all or too few
  • in any case, upon UV irradiation, solution volume significantly decreased
  • so I test a riboflavin gel with APS/TEMED top gel first ...

Comparison of a APS/TEMED and a riboflavin gel

We tested:

  • bovine serum albumine (BSA) protein mixture
  • Drosophila melanogaster nuclear protein extract

Result:

  • the APS/TEMED gel worked fine
  • the riboflavin-polymerized gel did not work:
  1. was fragile, fell apart upon transfer to coomassie staining solution
  2. homogenity problem: upper part okay, then some kind of disruption in the gel
  • we learned: illumination is critical
    • duration
    • intensity
    • homogenity

Conclusions

At the present state this protocol is not ready for every-day application in the lab. More experiments need to be carried out, to establish reproduceable results. We believe, a more intense, more homogenuous ultraviolet light-source would be required.

References

   Immediately before use, the large and small pore solutions were
degassed under vacuum to remove dissolved oxygen which would inhibit
the polymerisation, and one volume of a stock solution of 0.01 percent
riboflavin was added to 40 volumes of each.
They were supported a few centimetres away from a 25 watt fluorescent lamp.
Polymerisation was generally complete within 20 min and was accompanied
by the appearance of a slight opalescence in the gel and the formation
of a refractile boundary l-2 mm below the surface of the water layer.
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