Aug 22, 2020
NEXT Gel - CHEM 584
- 1Brigham Young University
Protocol Citation: Ken Christensen 2020. NEXT Gel - CHEM 584 . protocols.io https://dx.doi.org/10.17504/protocols.io.bj5dkq26
Manuscript citation:
Adapted from the NEXT Gel instructions included with the solution.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: August 22, 2020
Last Modified: August 22, 2020
Protocol Integer ID: 40837
Abstract
General Information: VWR Life Science AMRESCO’s NEXT GEL® products for denaturing gel electrophoresis are proprietary, ready-to-pour solutions comprised of acrylamide, bis-acrylamide, gel buffer, and SDS. The unique chemistry of NEXT GEL® eliminates the need for a stacking gel, thus reducing gel preparation time and extending the separation matrix available for electrophoresis, enabling the resolution of small peptides and high molecular weight proteins in the same gel.
NEXT GEL® solutions polymerize upon addition of ammonium persulfate and TEMED and are fully compatible with all standard electrophoresis equipment, SDS-PAGE staining procedures, and downstream applications including 2D electrophoresis, western blot, transfer, protein sequencing, and MALDI analysis. Each NEXT GEL® acrylamide solution is supplied with NEXT GEL® Running Buffer, 20X, which is essential for optimal gel performance.
Materials
MATERIALS
TEMEDBio-rad LaboratoriesCatalog #1610801
APSSigma AldrichCatalog #A-3678
NEXT Gel Acrylamide SolutionAmresco
NEXT Gel Running BufferAmrescoCatalog #M259
Safety warnings
Note: Acrylamide is a potent, cumulative neurotoxin that is absorbed through the skin. Always wear appropriate personal protective equipment, including gloves, when pouring and handling gels.
Prepare a fresh solution of 10% w/v ammonium persulfate in water. 1 mL is sufficient for many gels.
Add 3 µL of TEMED and 30 µL of freshly made 10% w/v ammonium persulfate (APS) to 5 mL of
NEXT Gel solution in a conical tube. Tighten cap and mix immediately by gently inverting. Pour between prepared glass plates, filling to the top.
Insert an appropriate comb and allow gel to polymerize for up to 00:30:00 minutes.
No stacking gel required!
Remove comb. Rinse wells with water to remove any residual gel pieces.
Assemble mini-gel system. Dilute NEXT GEL® Running Buffer, 20X to 1X by diluting 1:20 in deionized water. Prepare sufficient 1x NEXT Gel Running Buffer from a20X stock solution to fill both the anode and cathode chambers. For the Bio-Rad Mini-Gel Tetra System gel apparatus that we use in the lab, this means that you will need 350 mL of 1x Running Buffer for 1 gel and 700 mL of Running Buffer for 2 gels.
Prepare molecular weight markers and samples per standard preparation procedures and
load gel wells. Use the 6x SDS Loading Buffer for preparing samples.
Run the gel at 150 volts for up to 01:30:00 or until dye reaches
bottom of the gel.
Disassemble the gel apparatus and proceed with the downstream application.
Remove and stain gel for proteins using the Coomassie Blue staining solution for up to 01:00:00 or overnight. Transfer to nitrocellulose if performing a western blot.
For Coomassie Blue stained gels, destain using the Destain solution for up to 1h or to overnight. The addition of a Kimwipe to Destain can enhance the destaining process. Be careful not to destain too long as the protein bands will lighten.
FAQ's
FAQ's