Bis-Tris Handbook Merck
Bis-Tris Handbook Merck
Bis-Tris Handbook Merck
mPAGE™
Bis-Tris Precast SDS-PAGE Gels
Product Ordering.......................................................................................... 13
Notice........................................................................................................... 15
Contact Information..................................................................................... 15
Technical Assistance.................................................................................... 15
Standard Warranty....................................................................................... 15
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Introduction
The mPAGE™ Bis-Tris SDS-PAGE Gel system offers high performance, optimal electrophoretic separation, and
better resolution over a wide range of molecular weights. The Bis-Tris SDS-PAGE system helps preserve protein
integrity and extends the shelf life of the mPAGE™ Bis-Tris Precast Gel. mPAGE™ Bis-Tris Precast Gels have a
versatile design that allows for larger sample loading volumes. The 10 cm x 8 cm mini cassette format makes
mPAGE™ Bis-Tris Precast Gels compatible with most popular gel electrophoresis equipment.
mPAGE™ Bis-Tris Precast Gels are designed to work exclusively with MOPS or MES running buffer. Depending
on which running buffer is used, very distinct separation patterns can be achieved. MOPS buffer can be used to
fine tune the separation of large and medium-sized proteins, whereas MES buffer provides optimal separation of
smaller proteins. Refer to the migration charts (Figure 1) to determine which gel running buffer system is best
suited for the intended separation range.
The mPAGE™ Bis-Tris Precast SDS-PAGE Gel System includes a specially formulated transfer buffer optimized for
transferring proteins from mPAGE™ Bis-Tris Precast Gels to PVDF or nitrocellulose blotting membranes.
CAUTION: D
o not use Tris-glycine SDS running buffer with mPAGE™ Bis-Tris Precast Gels.
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Additional electrophoresis tanks listed below are compatible by using mPAGE™ Adapter Plates. Use one adapter
per gel. Position the adapters against the tall plate of the gel cassette before assembling gel tank. (Figure 5)
4. Fill the buffer core with 1X running buffer to check for a proper seal prior to filling the anode (outer) chamber
to the recommended level.
2. Heat samples for 10 minutes at 70 °C (Do not boil samples). Centrifuge samples prior to loading.
3. To load the samples into the wells, vertically insert tip for optimal sample loading results (Figure 6). Do not
exceed well capacity when loading samples: 80 μL for 10-well gels; 60 μL for 12-well gels; and 40 µL for
15-well gels.
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mPAGE™ MES SDS Running Buffer mPAGE™ MOPS SDS Running Buffer
180 V 200 V
Typical Amperage Typical Amperage
% Acrylamide Typical Run Time Typical Run Time
(Starting-Ending) (Starting-Ending)
8% 109-55 mA 22 min 103-47 mA 27 min
10% 105-50 mA 26 min 97-48 mA 31 min
12% 100-46 mA 30 min 97-42 mA 34 min
4-12% 107-53 mA 26 min 97-41 mA 36 min
4-20% 106-44 mA 40 min 87-40 mA 34 min
8-16% 113-48 mA 32 min 93-44 mA 31 min
Gel Staining
mPAGE™ Bis-Tris Precast Gels are compatible with popular gel staining protocols. When using commercially
available staining reagents, follow the manufacturer’s instructions.
Western Blotting
Gels perform best when using mPAGE™ Transfer Buffer for wet as well as semi dry transfer (see special
preparation instructions in semi-dry transfer section).
3. Submerge fiber pads in mPAGE™ Transfer Buffer containing 10% methanol and remove air bubbles. Place the
appropriate number of pads onto the blot module cathode plate.
4. Open the gel cassette. (See Figure 7). To maintain consistent orientation, carefully remove the short plate,
allowing the gel to remain on the tall plate. Remove the stacker by cutting 5 mm below the well bottom.
5. Place one prewetted piece of filter paper on top of the gel. Using a roller or serological pipette, remove any air
bubbles.
6. Turn the tall plate over, holding over the removed short plate (or gloved hand), carefully separate the gel from
the tall plate.
7. Transfer the gel/filter paper assembly onto the fiber pad with the gel facing up and the filter paper contacting
the fiber pads. Add a small amount of mPAGE™ Transfer Buffer on the gel before placing the blotting
membrane. Using a roller or serological pipette, remove any air bubbles between gel and membrane.
8. Place a second piece of prewetted filter paper on top of the membrane. Using a roller or serological pipette,
remove any air bubbles.
9. Place an additional fiber pad(s) on top of the filter paper. Close the assembly and place into the
electrophoresis tank. Due to differences in transfer systems refer to blot module user guide for further
instructions.
10. Connect tank to a power supply and transfer as outlined in the blot module instructions. Depending on
molecular weight of the protein of interest, further optimization of transfer time may be required.
See Table 3 for examples of typical transfer times for popular wet transfer systems.
Table 3. Popular Wet Transfer Systems
11. Remove the blot from the blot module and rinse the membrane in deionized water to remove transfer buffer
and residual gel debris.
12. To visualize the transferred proteins prior to immunodetection, the membrane may be stained with any
reversible blot stain compatible with immunodetection. Follow the reagent manufacturer’s staining protocol.
13. The blot may be dried or used immediately in a desired immunodetection protocol.
1. Prepare 10X mPAGE™ Transfer Buffer stock solution by dissolving 1 packet of mPAGE™ Transfer Buffer in
100 mL deionized water.
2. Prepare 100 mL 2X mPAGE™ Transfer Buffer containing 10% methanol:
• Methanol: 10 mL
• Deionized Water: 70mL
• 10X mPAGE™ Transfer Buffer Stock Solution
Note: If transferring high molecular weight proteins, buffer may be supplemented with 0.025-0.05% SDS.
Buffer Formulations
Table 4. mPAGE™ 4X LDS Sample Buffer Table 7. mPAGE™ 1X Transfer Buffer
pH 8.2 for Wet Transfer protocol
Reagent Amount
Tris-HCl 0.666 g Reagent concentration Amount
Tris-Base 0.682 g 25 mL Tris base 3.0 g
Lithium dodecyl sulfate (LDS) 0.800 g 25 mM Bicine 4.08 g
EDTA 0.006 g 10% Methanol 100 mL
Glycerol 4g Deionized water 900 mL
Coomassie® Brilliant Blue G250 (1% solution) 0.75 ml
Phenol Red (1% solution) 0.25 ml Table 8. mPAGE™ Transfer Buffer (with Methanol)
Deionized water To 10 ml pH 8.2 for Semi Dry Transfer protocol
Reagent Amount
Tris-Base 6.06 g
MOPS 10.46 g
SDS 1.0 g
EDTA 0.3 g
Deionized water 1000 mL
The information in this document is subject to change without notice and should not be construed as a
commitment by the manufacturing or selling entity, or an affiliate. We assume no responsibility for any errors that
may appear in this document.
Contact Information
For the location of the office nearest you, go to SigmaAldrich.com/offices.
Technical Assistance
Visit the tech service page on our web site at SigmaAldrich.com/techservice.
Standard Warranty
The applicable warranty for the products listed in this publication may be found at SigmaAldrich.com/terms.
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