Monitoring protein migration during SDS-polyacrylamide gel electrophoresis.
Monitoring protein transfer onto membranes after Western blotting.
Sizing of proteins on SDS-polyacrylamide gels and Western blots.
Description
Spectra™ Multicolor Broad Range Protein Ladder is designed for monitoring protein separation during SDS-polyacrylamide gel electrophoresis, verification of Western transfer efficiency on PVDF, nylon and nitrocellulose membranes and for approximate sizing of proteins (1-3). It is composed of prestained recombinant prokaryotic proteins.
Spectra™ Multicolor Broad Range Protein Ladder is a 4-color protein standard with 10 prestained recombinant prokaryotic proteins covering a wide range molecular weights from 10 to 260 kDa. Four different chromophores are bound to the proteins producing a brightly colored ladder with an easy-to-remember pattern.
Storage Buffer
62.5 mM Tris-H3PO4 (pH 7.5 at 25°C), 1 mM EDTA, 2% SDS, 10 mM DTT, 1 mM NaN3 and 33% glycerol.
Quality Control
Tested in SDS-polyacrylamide gel electrophoresis and Western Blotting.
Storage
Stable at 4°C for up to 3 months. For long term storage, store at -20°C.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Low percentage gels are recommended for analysis of large proteins and high percentage gels for analysis of small proteins.
Linear gradient gels allow for high resolution of a broad range of both small and large proteins. Gel Recommendations.
Protein MW range, kDa
Recommended gel, %
~5-50
18
~5-60
16
~10-80
14
~20-150
12
~30-200
10
~40-250
8
~60-300
6
~100-400
4
Protein MW range, kDa
Recommended gradient gel, %
~5-100
10-20
~5-300
4-20
~10-200
8-16
~30-300
4-12
All Fermentas protein ladders/markers can be used on 6, 8, 10, 12, 14 % SDS polyacrylamide gels and on 4-12%, 8-16%, 4-20% and 10-20% gradient gels.
The following general rule can be applied to protein ladders/markers as well as to protein samples:
in low percentage gels (4-8%), small proteins (10-15 kDa) migrate with the tracking dyes during electrophoresis and may be not visible;
in high percentage gels (14-18%) large proteins (150-250 kDa) may not separate.
For more precise determination of molecular weights, unstained protein ladders/markers are recommended.
Prestained standards are ideal for monitoring the process of electrophoresis and the protein transfer efficiency in Western blotting.
Prestained proteins may have different mobilities in various SDS-PAGE buffer and gel systems due to coupled chromophores that affect protein mobility. Prestained standards are recommended when approximate sizing is enough.
Each lot of prestained protein ladder/marker is calibrated against a precisely sized unstained protein ladder/marker in Tris-glycine-SDS gel and the calculated apparent molecular weights are reported in the product's Certificate of Analysis. The prestained protein may have different mobility in other electrophoresis buffer and gel systems.
Modifications to native proteins such as phosphorylation and glycosylation may alter protein mobility. The molecular weights of modified proteins may not correspond to those of unmodified proteins of the same size.
Protein Ladder/Marker: Recommendations for Loading
Thaw the ladder either at room temperature or at 37°C for a few minutes to dissolve precipitated solids. Do not boil.
Mix gently, but thoroughly, to ensure that the solution is homogeneous.
For Unstained Protein Molecular Weight Marker only:
transfer the required aliquot to a clean tube with a screw cap;
heat at 95°C for 10 minutes;
cool and mix. Once denatured the marker can be further used just after the thawing.
Load the following volumes of the ladder/marker on SDS-polyacrylamide gel with a thickness of 0.75 mm:
5 µl per well for mini-gels;
10 µl per well for large gels. For Spectra™ Multicolor Broad Range Protein Ladder:
10 µl per well for mini-gels;
20 µl per well for large gels.
Note
To avoid overloading the gel which will be subsequently silver stained, dilute the ladder/marker just prior to use: Water, nuclease-free: 36.5 µl 5X Protein Loading Buffer*: 10 µl 20X Reducing Agent (#R0891): 2.5 µl Protein ladder/marker: 1 µl
* Alternatively 4X DualColor™ Protein Buffer Loading Pack can be used. Volumes of the buffer and water should be adjusted appropriately.
Staining is not required to visualize prestained protein ladders/markers. To visualize unstained protein ladders/markers, the gel can be processed with PageBlue™ Protein Staining Solution, PageSilver™ Silver Staining Kit or other protein staining techniques. For silver staining, the volume of Unstained Protein Molecular Weight Marker used should be decreased up to 10-fold.
Protein Samples: Preparation for Loading on SDS-PAGE
Step
Sample preparation with the DualColor™ Protein Loading Buffer Pack
Sample preparation with the Protein Loading Buffer Pack
Thaw
Thaw the buffer pack components either at room temperature or at 37°C for a few minutes to dissolve precipitates.
Mix
Vortex gently, but thoroughly to ensure that the solution is homogeneous.
Dilution
2.0 µl of 20X Reducing Agent
2.5 µl of 20X Reducing Agent
Protein sample (~0.5 ng – 2.5 µg) For Western blots or gels to be treated with Coomassie based stains, use up to 2.5 µg of total protein per minigel well. For silver staining applications use up to 10 ng of total protein per minigel well.
10 µl of 4X DualColor™ Protein Loading Buffer Water, nuclease-free to 40 µl*
10 µl of 5X Protein Loading Buffer Water, nuclease-free to 50 µl*
Denaturation
Incubate samples at 95-100°C for 5 minutes.
Loading
Spin for a few seconds in a microcentrifuge. Apply directly to an SDS-polyacrylamide gel. Use ~10 µl per minigel well.
* The sample volume can be scaled up or down.
Semi-dry Protein Transfer for Western Blotting
Note
Wear gloves throughout the procedure to avoid contamination. Use 100 ml of each solution for mini gels (8x10 cm; 10x10 cm), for larger gels use enough of the solution to completely cover the gel/membrane/paper sheets in each step. Buffers Tris-glycine-methanol protein transfer buffer. Cool at 4°C before use.
Component
Amount
Final concentration
10X Tris-glycine buffer
10 ml
1X
Methanol
10 ml
10% (v/v)
Deionized water
to 100 ml
CAPS buffer for electrotransfer of proteins onto PVDF for N-terminal sequencing. Cool at 4°C before use.
Component
Amount
Final concentration
10X CAPS (100 mM, pH 11.0)
10 ml
10 mM
Methanol
10 ml
10% (v/v)
Deionized water
to 100 ml
10X CAPS (3-[cyclohexylamino]-1-propanesulfonic acid). Store at 4°C.
Component
Amount
Final concentration
CAPS
2.21 g
100 mM
Deionized water
to 90 ml
2N NaOH
titrate to pH 11.0 (~4 ml)
Deionized water
to 100 ml
Ponceau S staining solution (only freshly made staining solution should be used).
Component
Amount
Final concentration
Ponceau S
0.2 g
0.2% (w/v)
Glacial acetic acid
1 ml
1% (v/v)
Deionized water
to 100 ml
Semi-dry Protein Transfer
Presoak 2-4 pieces of blotting paper (cut to the size of the gel) in transfer buffer for 5 min.
Cut a piece of nitrocellulose membrane to the size of the gel and equilibrate it in transfer buffer. If a PVDF membrane is used, incubate it in methanol for 2 min before equilibrating it in transfer buffer. Use CAPS buffer for N-terminal sequencing, Tris-glycine-methanol protein transfer buffer is suitable for all other applications.
Carefully remove the stacking gel from the resolving gel. Soak the resolving gel in CAPS buffer for 5 min if this buffer is used. This step can be omitted if Tris-glycine-methanol protein transfer buffer is used.
Assemble the transfer sandwich with the resolving gel on the anode (+) as shown in Figure below. Use one sheet of blotting paper or two pieces of filter paper on each side of the sandwich. Make sure all air bubbles are removed since they will affect the efficiency of the electroblotting.
Electrotransfer proteins from the gel on the membrane for ~60 min at room temperature. Maintain the current at 0.8 mA per 1 cm2 of the gel area and limit the voltage to 15 V.
When the transfer is complete, turn off the power and peel off the layers of the sandwich until you reach the membrane. Remove the membrane with forceps and rinse it in deionized water.
Monitoring the Protein Transfer The efficiency of electrotransfer can be monitored using prestained protein ladders (Spectra™ Multicolor Broad Range Protein Ladder, PageRuler™ Prestained Protein Ladder, PageRuler™ Plus Prestained Protein Ladder and Prestained Protein Molecular Weight Marker. The use of the DualColor™ Protein Loading Pack also allows for monitoring of Western blot protein transfer from gel to membrane. Alternatively, the extent of protein transfer can be determined by staining the polyacrylamide gel after the transfer or by staining the protein directly on the membrane. Proteins on PVDF membranes can be visualized with the PageBlue™ Protein Staining Solution, while Ponceau S, India Ink or Amido Black are recommended for nitrocellulose and PVDF membranes. Figure. Blotting with a semi-dry transfer unit.
Migration Patterns in Different Electrophoresis Conditions
Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680-685, 1970.
Burnette, W.N., “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate – polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A, Anal. Biochem., 112 (2), 195-203, 1981.
Towbin, H., et al., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA, 76, 4350-4354, 1979.
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- Store at -20°C
