Mini SDS-PAGE Gel Protocol for the Novex Surelock Cell

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Mini SDS-PAGE Gel Protocol for the Novex Surelock Cell
Note: For best results, use MilliQ or nanopure equivalent water to prepare all buffers and
samples.
Preparing 1X NuPAGE SDS PAGE Running Buffer
Components
20X NuPAGE MOPS or MES Running Buffer
Nanopure water
Amount for 0.8 L
40 ml
760 ml
The 1X can be made several days in advance.
Note: If the 20X or 1X buffer is turning yellow, the glycine is degrading and so it is time to properly
dispose of it.
Sample Preparation
1) Prepare the samples:
10-Well Gels-maximum loading volume 25 µl/well (1.0 mm thick) & 37 µl/well (1.5 mm thick)
12-Well Gels-maximum loading volume 20 µl/well (1.0 mm thick)
15-Well Gels- maximum loading volume 15 µl/well (1.0 mm thick) & 25 µl/well (1.5 mm thick)
Sample
Water
NuPAGE LDS Sample Buffer 2.5 μl
(4X)
NuPAGE Reducing Agent (10X)* 1.0 μl
Total Volume
10 μl
2) Prepare 1X sample buffer to fill the empty wells:
Water
NuPAGE LDS Sample Buffer (4X)
NuPAGE Reducing Agent (10X)*
Total Volume
3.75 μl
5 μl
6.25 μl
10 μl
1.5 μl
15 μl
2.0 μl
20 μl
2.5 μl
25 μl
4.0 μl
40 μl
650 μl
250 μl
100 μl
1000 μl
Note: *Add the Reducing Agent within 1 hr of loading. Do not freeze sample after adding.
Reoxidation of samples occur during storage and produce inconsistent results.
3) Heat the samples at 70°C for 10 minutes for optimal results. Do not boil your samples. Boiling the
sample at 100°C in SDS containing buffer results in proteolysis (Kubo, K. Anal Biochem 1995, 351353).
Note for people who are working with membrane proteins: Do not boil your sample because it
may cause your proteins to aggregate. Instead, load your sample immediately after adding the
loading buffer.
1
Preparing a Precast Gel for Electrophoresis
1)
2)
3)
4)
5)
Remove the precast gel from the storage pouch.
Rinse the outside of the gel thoroughly with distilled water.
Remove the tape from the slot on the back of the gel cassette.
In one fluid motion, gently remove the comb.
Rinse the wells thoroughly with either: distilled water or 1X running buffer. Invert the gel and shake to
remove excess buffer/water. Repeat twice. If necessary, straighten the sides of the wells.
Assembly of the Surelock Cell
1) Lower the Buffer Core into the Lower Buffer Chamber so that the negative electrode fits into the
opening in the gold plate on the Lower Buffer Chamber.
2) Insert the Gel Tension Wedge into the cell behind the buffer core. Make sure the Wedge is in the
unlock position.
3) Insert gel cassettes into the Lower Buffer Chamber by place one cassette in front of the Buffer Core.
Place the other gel or Buffer Dam behind the Buffer Core. The shorter plate/”well” side of the cassette
needs to face towards the Buffer Core (inside). The slot at the bottom of gel must face towards the
buffer in the Lower Buffer Chamber (outside).
4) Pull forward on the Gel Tension Lever until the lever comes to a firm stop.
Pull lever this direction.
5) Fill the upper/inner chamber with 200 ml of the appropriate Running Buffer. Make sure there is
enough buffer to completely cover the sample wells. Check to see that the buffer is not leaking from
the inner chamber into the outer chamber.
6) Add 500 μl of the NuPAGE Antioxidant to upper/inner chamber buffer just prior to loading your
samples. Mix thoroughly. (
Note: The Antioxidant prevents sample reoxidation and maintains the proteins in a reduced
state. In the neutral pH environment of NuPAGE gels, DTT and β-mercaptoethanol tend to
remain at the top of the gel, and do not co-migrate with the sample. The Antioxidant migrates
with the proteins during electrophoresis, and protects disulfide bonds and sensitive amino
acids (e.g. methionine and tryptophan) from oxidizing.
Sample Loading
1) Using a gel loading pipette tip, load the molecular weight standards (usually in lane 1).
Note: All Molecular Weight Standards are “not” compatible with the NuPAGE gels. Use only the
standards listed below in the Supply Table or ones approved by Life Technologies.
2
2) Load your samples into the appropriate wells by lowering the pipette tip to the bottom of the sample
well and slowly pipet the sample into well. Be careful not to contaminate another well with the sample
or puncture the bottom of the well.
3) Load 20 µl 1X Sample loading buffer into any remaining empty wells. This promotes uniform running of
the stacking front.
Running Gel(s)
1) Fill the Lower Buffer Chamber with the remaining 600 ml of Running Buffer by pouring it in the gap
between the Gel Tension Wedge and the back of the Lower Buffer Chamber.
2) Place the lid on the Mini-Cell. Make sure the lid is properly seated or no power will go through the MiniCell.
3) With the power off, insert the electrical leads into the lead adapters in the Bio-Rad PowerPac HC.
Caution: You must use HV lead adapters when using the Bio-Rad power supply with the Novex
Surelock Mini-Cell; otherwise you will cook your gel.
HV Lead Adapters
4) Apply power to the Mini-Cell and begin electrophoresis:
Standard Method
200 V until the dye front reaches the bottom
Low Voltage Method-Membrane Proteins and Bands for Mass Spectrometry Analysis
40 V for 10 min (allows better stacking of the proteins)
100 V until the dye front reaches the bottom
Note: The low voltage method reduces the amount of heat which is generated during
electrophoresis resulting in sharper bands. For further cooling, the gel maybe ran in a
coldroom/coldbox. Don’t prechill the running buffer since colder temperatures will cause
the SDS to come out of solution.
Gel Removal
1)
2)
3)
4)
At the completion of the run, turn off the power supply and disconnect the electrical leads.
Remove the tank lid and unlock the Gel Tension Lever.
Remove the gel cassettes from the Mini-Cell.
Lay the gel with the bottom slot/long plate facing down. Insert the Gel Knife into the gap between the
two plastic plates to separate each of the three bonded sides of the cassette.
5) Gently push down on the Gel Knife to separate the plates. You will hear a cracking sound which
means you have broken the bonds which hold the plates together. Repeat on each side of the cassette
until the plates are completely separated.
Caution: Be careful while inserting the Gel Knife between the gel plates or you may cut into the
gel.
3
6) Carefully remove the top plate. The gel may adhere to either side. Discard the plate without the gel.
7) Hold the cassette plate over a container with water or transfer buffer with the gel side facing towards
the container.
If the gel is stuck to the plate with the bottom slot, use the Gel Knife to push the gel foot through the
slot. The gel should be in contact with the fluid, and with some with gentle agitation, the gel should fall
into the fluid.
If the gel is stuck to the short plate, place the gel into the fluid and agitate gently until the gel separates
from the plate. You may need to use Gel Knife to carefully loosen the lower corner of the gel and then
allow the gel to peel away from the plate.
Note: If the gel will be transferred, the gel foot will need to be cut off with a Gel knife.
8) Discard the running buffer into the proper hazardous waste container.
9) Rinse the Mini-Cell and all its components with distilled, deionized water.
References
The protocol above was based in part, on the following literature from Life Technologies:
• XCell SureLock Mini-Cell Manual (MAN0000739, Rev. Date 6 May 2010)
• NuPAGE Technical Guide (MAN0003188, Rev. Date 29 October 2010)
Written by Melissa Sondej (version 4/17/12)
4
Supplies
Below lists the supplies needed.
Gels
NuPAGE 4-12% Bis-Tris Gel, 10 well, 1.0 mm, 25 µl
NuPAGE 4-12% Bis-Tris Gel, 10 well, 1.5 mm, 37 µl
NuPAGE 10% Bis-Tris Gel, 10 well, 1.0 mm, 25 µl
NuPAGE 12% Bis-Tris Gel, 10 well, 1.0 mm, 25 µl
NuPAGE 4-12% Bis-Tris Gel, 12 well, 1.0 mm, 20 µl
NuPAGE 10% Bis-Tris Gel, 12 well, 1.0 mm, 20 µl
NuPAGE 12% Bis-Tris Gel, 12 well, 1.0 mm, 20 µl
NuPAGE 4-12% Bis-Tris Gel, 15 well, 1.0 mm, 15 µl
NuPAGE 4-12% Bis-Tris Gel, 15 well, 1.5 mm, 25 µl
* Gels are also sold in a 10 pack.
Reagents
NuPAGE MOPS SDS Running Buffer (20X)
NuPAGE MOPS SDS Running Buffer (20X)
NuPAGE Sample Reducing Agent (10X)
NuPAGE Antioxidant
NuPAGE LDS Sample Buffer
Protein Standards
BenchMark Unstained Protein Ladder (Sypro Ruby)
Novex Sharp Unstained Protein Standard (Sypro Ruby)
Novex Sharp Prestained Protein Standard (Western)
Stain
Sypro Ruby Fluorescent Protein Stain (10 ng sensitivityovernight)
Gelcode Blue Stain Reagent
(25 ng sensitivityovernight)
Homemade Coomassie Blue (100 ng sensitivity)
Imperial Protein Stain (>10 ng sensitivity)
Size
Vendor
Catalog Number
2 gels
2 gels
2 gels
2 gels
2 gels
2 gels
2 gels
2 gels
2 gels
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Life Technologies
NP0321PK2
NP0335PK2
NP0301PK2
NP0341PK2
NP0322PK2
NP0302PK2
NP0342PK2
NP0323PK2
NP0336PK2
500 ml
500 ml
250 μl
15 ml
10 ml
Life Technologies
Life Technologies
Life Technologies
Life Technologies
Life Technologies
NP0001
NP0002
NP0004
NP0005
NP0007
2 x 250 µl
2 x 250 µl
2 x 250 µl
Life Technologies
Life Technologies
Life Technologies
10747012
LC5801
LC5800
200 ml
Life Technologies
S12001
500 ml
Thermo Scientific
Pierce
24590
200 ml
Thermo Scientific
Pierce
Bio-Rad
161-0495
2.5 L
4L
EMD/VWR
EMD/VWR
EMD-AX0073-9
EM-MX0475-1
1L
Oriole Fluorescent Gel Stain
Fixer/Destain Solution
Acetic Acid
Methanol
5
24615
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