Amresco 1-2
Next Gel TM Electrophoresis System
1. Gather reagents needed:
a. NEXT GEL TM Kit:
i. NEXT GEL TM solution, 1X
1. acrylamide, bisacrylamide, SDS, gel buffer.
ii. NEXT GEL TM Running Buffer, 20X
1. Do not use other running buffers with the NEXT GEL TM
system.
2. Buffers not formulated for NEXT GEL TM will introduce
artifacts that impair band resolution.
b. Required reagents not included in kit:
i. TEMED
ii. Ammonium Persulfate (APS)
iii. Sample loading buffer
2. Prepare gel solution.
a. Since stacking gels are not used with NEXT GELTM it is necessary to
prepare sufficient NEXT GELTM solution to equal the total volume of a
traditional resolving gel plus the stacking gel.
3. For a 10 cm x 10 cm x 0.75 mm mini-gel, pour 10 ml of NEXT GELTM solution
into a conical tube.
4. Add 60 ul of APS/TEMED Polymerization Tablet stock solution OR 60 ul of
freshly made 10% Ammonium Persulfate and 6ul of TEMED
5. Tightly cap the tube and gently invert the solution to mix
a. Do Not Vortex.
6. Immediately pour the solution between the glass plate.
a. If the NEXT GELTM solution is at room temperature it is not necessary to
de-gas prior to pouring the gel.
b. The solution should be poured to the top of the plates since stacking gels
are not used with the NEXT GELTM system.
7. Immediately insert comb and allow gel to polymerize completely, about 10 to 30
minutes.
a. The 5% NEXT GELTM (M254) may take up to 1 hour to polymerize.
8. Remove comb and rinse well with water or running buffer to remove any residual
gel pieces.
9. Drain wells completely.
10. Assemble gel system and completely fill both anode and cathode chambers with
sufficient 1X NEXT GELTM Running Buffer
a. Diluted from the supplied 20X stock solution.
b. Please refer to the operations manual for your specific apparatus for
volume recommendations.
c. For optimal resolution use only the supplied NEXT GELTM Running
Buffer at the recommended 1X solution
d. Do not use other running buffers with the NEXT GELTM system.
e. Buffers not formulated for NEXT GELTM will introduce artifacts that
impair band resolution.
Amresco 2-2
11. Sample Preparation:
a. Final protein concentration should be about 0.16 -10.0 ug/ul for a
heterogenous mixture and about 0.02 – 0.1 ug/ul for purified proteins.
b. Electrophoresis on NEXT GELTM is sensitive to the amount of protein
loaded on the gel.
c. Protein overloading or high concentrations of salts, lipids of nucleic acids
can increase electrical resistance that will overheat gels.
d. Protein overloading can lengthen the run time and generate band distortion.
e. To optimize results on mini-gels load about 0.2 ug – 1.0 ug per band per
lane for Coomassie Blue staining.
f. For complex protein mixtures such as cell lysates, load about 1.6 ug – 100
ug of protein per lane.
g. Reduce the amount of protein 10 to 100 fold for silver staining.
h. Reduce the concentrations of salt, lipids and nucleic acids in the loading
sample to reduce resolution and generate band distortion.
12. Dilute 1 part 4X Sample Loading Buffer (M260) with 3 parts sample.
13. Boil 3-5 minutes in water bath and cool.
14. Load 10-20 ul per well for minigels.
a. Conventional loading buffers such as Amresco 2X Protein Loading Buffer
of Laemmli sample buffer may be used according to standard procedures.
15. Run gel at 150 volts for 60 to 90 minutes or until tracking dye reaches bottom of gel.
a. When switching to the NEXT GELTM monitor initial runs to ensure that
voltage remains constant.
16. Disassemble the apparatus
17. Allow gel to cool briefly before removing from plates.
18. Remove gel and stain with standard methods.
19. NEXT GELTM can be stained with all common SDS-PAGE procedures and is fully
compatible with downstream applications including 2D electrophoresis, Western
blot transfer protein sequencing and MALDI analysis.
Caution:
Acrylamide:
Potent, cumulative neurotoxin that is absorbed through the skin.
Always wear gloves when pouring and handling gels.
20X Next GelTM Running Buffer:
Irritant due to the high salt concentration.
The pH is neutral.
Rinse with water if spilled on skin.