Introduction. Protein electrophoresis is a method of analysing a mixture of proteins by means of gel electrophoresis. Fermentas offers products for native protein extraction, quantification and analysis, that are the fundamental steps in proteomics research. More>>
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Ready-to-use – supplied in a loading buffer for direct loading on gels.
Sharp bands.
Contains a 25 kDa reference band of greater intensity.
Ladder proteins contain an integral Strep-tag® II sequence.
Applications
Accurate molecular weight determination of polypeptides in SDS-PAGE and Western blots.
Description
PageRuler™ Unstained Low Range Protein Ladder is designed for accurate sizing of small proteins by SDS-PAGE. It is a mixture of 7 recombinant, highly purified proteins, ranging in size from 5 to 100 kDa and a synthetic 3.4 kDa peptide. The proteins resolve into clearly identifiable sharp bands when analyzed by SDS-PAGE and stained with Coomassie Blue or silver (1, 2). The 100 kDa protein band indicates the approximate position of large proteins and the 25 kDa band is of greater intensity and serves as a reference band.
Proteins can be detected on Western blots by staining with Ponceau S, Coomassie Blue or other protein stains (2, 3). In addition, the ladder proteins (except for the 5 and 3.4 kDa peptides) contain an integral Strep-tag II sequence and may be detected on Western blots using Strep-Tactin® conjugates (4).
The PageRuler™ Unstained Low Range Protein Ladder is supplied ready-to-use: no heating, dilution or addition of a reducing agent is required before use.
Content
The PageRuler™ Unstained Low Range Protein Ladder is provided in the following storage buffer: 62.5 mM Tris-H3PO4 (pH 7.5 at 25°C), 1 mM EDTA, 2% (w/v) SDS, 0.1 M DTT, 1 mM NaN3, 0.01% (w/v) bromophenol blue and 33% (v/v) glycerol.
Quality Control
Tested in SDS-polyacrylamide gel electrophoresis and Western blotting.
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.
General Protocol for SDS-PAGE
I. Reagents 30% acrylamide/bisacrylamide (37.5:1) aqueous solution (stored in the dark) 1.5 M Tris-HCl buffer (pH 8.8) 0.5 M Tris-HCl buffer (pH 6.8) 10% ammonium persulfate (APS) solution (always should be prepared freshly) TEMED 1X Tris-glycine-SDS Buffer (10X buffer diluted to 1X concentration prior use) Caution. Acrylamide is a neurotoxin. Always wear gloves, safety glasses, and a surgical mask when working with acrylamide powder. II. Resolving Gel Preparation
Components
Volume: 10 ml resolving gel solution (for 2 minigels)
for 8% gel
for 10% gel
for 12% gel
Deionized water
4.73 ml
4.13 ml
3.43 ml
30% acrylamide/bisacrylamide
2.7 ml
3.3 ml
4.0 ml
1.5 M Tris-HCl containing 0.4% SDS, pH 8.8
2.5 ml
2.5 ml
2.5 ml
10% APS
60 µl
60 µl
60 µl
TEMED
13 µl
13 µl
13 µl
III. Stacking Gel Preparation
Components
Volume: 5 ml stacking gel solution (for 2 minigels)
Deionized water
3.0 ml
30% acrylamide/bisacrylamide
700 µl
0.5 M Tris-HCl containing 0.4% SDS, pH 6.8
1.25 ml
10% APS
25 µl
TEMED
20 µl
IV. Procedure
Assemble the glass plate sandwich. Prepare the resolving gel solution as described above.
Add APS and TEMED last, mix carefully to avoid formation of bubbles.
Important Note. Polymerization begins as soon as APS is added to the mixture, so all subsequent actions must be performed promptly.
Pour the gel solution between the glass plates with a pipette, leave about 1/4 of the space free for the stacking gel. Carefully cover the top of the resolving gel with 50% isopropanol, 0.1% SDS solution or water, and wait until the resolving gel polymerizes (~30 min). A clear line will appear between the gel surface and the solution on top when polymerization is complete.
Discard the water, isopropanol or SDS solution. Wash gently with double-distilled water.
Pour the stacking gel solution (prepared as described above, add APS and TEMED last) carefully with a pipette to avoid formation of bubbles. Important Note. Polymerization begins as soon as APS is added to the mixture, so all subsequent actions must be performed promptly.
Insert combs. Allow the gel to polymerize for at least 60 min.
Remove combs carefully. Put the gel into the electrophoresis tank, fill the tank (bottom and top reservoirs) with fresh 1X Tris-glycine-SDS Buffer, make sure that the gel wells are covered with the buffer.
Load protein ladder/marker and probes.
Set an appropriate voltage and current depending on how many gels you run. Increase the power when the dye front reaches the running gel. For exact values refer to the table below:
Gel
1 minigel
2 minigels
Stacking gel (upper)
13 mA
25 mA
Resolving gel (lower)
25 mA
50 mA
Values presented are for 0.75 mm gels. For thicker gels the current should be appropriately increased.
Stop the electrophoresis run when the dye front reaches the bottom of the gel. Disassemble the gel sandwich and proceed with gel staining or Western blot procedures.
Gel Staining Procedure with PageBlue™ Protein Staining Solution
With microwaving (fast protocol)
Without microwaving (conventional protocol)
Total time
25 min for native gels 40 min for SDS-containing gels
65 min for native gels 95 min for SDS-containing gels
1. Washing in water*. Repeat 3 times
Add 100 ml water
Microwave for 1 min
Wash with gentle agitation for 5 min
Discard the water
Add 100 ml water and rinse
Discard the water
2. Staining
Add 20 ml PageBlue™ Protein Stainin Solution
Microwave for 30 s
Stain with gentle agitation for 20 min
Discard the solution
Add 20 ml PageBlue™ Protein Staining Solution
Stain with gentle agitation for 60 min (or overnight)
Discard the solution
3. Washing in water
Add 100 ml water and rinse for 5 min
Add 100 ml water and rinse for 5 min
* only SDS-containing gels
Note
PageBlue™ Protein Staining Solution can be reused up to 3 times without a decrease in sensitivity.
All reagent volumes are for 8x10 or 10x10 cm minigels of 0.75-1 mm thickness. Gels should be completely immersed in solution.
When several gels are being stained, increase the amount of staining solution accordingly.
The first wash step is crucial to remove SDS from the gel as SDS interferes with the staining reaction.
For staining native gels without SDS, the washing step is not required.
Staining sensitivity can be increased if the proteins are fixed for 15 min either with 12% trichloracetic acid or with 25% isopropanol supplemented with 10% acetic acid. Fixation prevents protein diffusion from the gel and accelerates SDS removal. After fixation, gels can be stained immediately without additional washing.
Using either the fast or conventional protocol, staining sensitivity is 5 ng of protein per band. To increase sensitivity to 0.05 ng per band the gel can be stained using the PageSilver™ Silver Staining Kit.
For staining peptides or small proteins (more than 10 kDa) fixation of the proteins for 15 min either with 12% trichloracetic acid or with 25% isopropanol supplemented with 10% acetic acid is recommended. Fixation prevents protein diffusion from the gel and accelerates SDS removal. After fixation, gels can be stained immediately without additional washing. Overnight staining time is required for peptide detection.
Gel Staining Procedure with PageSilver™ Silver Staining Kit
With microwaving (fast protocol)
Without microwaving (conventional protocol)
Total time Sensitivity
1 hour 0.1 ng/band
2 hours 40 min 0.05 ng/band
1. Gel fixing 1
Rinse the gel with deionized water
Add gel fixing solution 1
Microwave for 30 s. Do not boil
Fix with gentle agitation for 10 min
Discard the solution
Rinse the gel with deionized water
Add gel fixing solution 1 and gently agitate for 60 min
Discard the solution
2. Gel fixing 2 and Washing
Perform Gel fixing 2 procedure twice:
Add gel fixing solution 2
Microwave for 30 s. Do not boil
Fix with gentle agitation for 10 min
Discard the solution
Perform Washing procedure twice:
Add deionized water and gently agitate for 20 s
Discard the water
Perform Gel fixing 2 procedure three times:
Add gel fixing solution 2 and gently agitate for 20 min
Discard the solution
Perform Washing procedure twice:
Add deionized water and gently agitate for 20 s
Discard the water
3. Sensitizing and Washing
Add sensitizing solution and gently agitate for 1 min
Discard the solution
Perform Washing procedure twice:
Add deionized water and gently agitate for 20 s
Discard the water
Add sensitizing solution and gently agitate for 1 min
Discard the solution
Perform Washing procedure twice:
Add deionized water and gently agitate for 20 s
Discard the water
4. Staining and Washing
Add staining solution and gently agitate for 20 min
Discard the solution
Perform Washing procedure twice:
Add water and gently agitate for 20 s
Discard the water
Add staining solution and gently agitate for 20 min
Discard the solution
Perform Washing procedure twice:
Add water and gently agitate for 20 s
Discard the water
5. Developing
Add developing solution and gently agitate for ~4 min
Discard the solution
Add developing solution and gently agitate for 5-10 min
Discard the solution
6. Terminating
Add stop solution and gently agitate for 5 min
Discard the solution
Add stop solution and gently agitate for 10 min
Discard the solution
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.
Staining PVDF Membrane with PageBlue™ Protein Staining Solution
Note
PVDF membrane must be air-dried before staining.
Add PageBlue™ Protein Staining Solution to cover the PVDF membrane. Agitate gently for 2 min.
Wash with 30% ethanol with gentle agitation for 5 min.
To completely remove the stain, wash the membrane with the mixture of 30% acetonitrile and 20% ethanol for 5 min.
SDS-PAGE for Small Proteins
We recommend to use special electrophoresis conditions that improve the resolution of small peptides (1 to 20 kDa). The main differences compared to a conventional gel include:
higher Tris concentration in gel buffer (0.75 M
instead of 0.375 M);
pH of 8.45 for both stacking and resolving gels;
higher acrylamide cross-linking in resolving gel (C= 5% instead of the usual ~3%);
ethylene glycol is included in the resolving gel.
Reagents
Ethylene glycol
Stock solutions of acrylamide/bisacrylamide 19:1 and 29:1
3 M Tris-HCl buffer containing 0.4% SDS, pH 8.45
40% ammonium persulfate (APS)
TEMED
1X Tris-tricine-SDS Buffer (10X Buffer (#B48), diluted to 1X concentration prior to use)
Gel Preparation The protocol is sufficient for two 0.75 mm mini gels.
Component
5% Stacking gel (C=3.3%)
18% Resolving gel (C=5%)
Ethylene glycol
–
2.4 ml
3 M Tris-HCl buffer, pH 8.45
1 ml
2 ml
Acrylamide/bisacrylamide (40%)
0.5 ml (29:1)
3.6 ml (19:1)
Deionized water
2.5 ml
–
40 % APS
4 μl
8 μl
TEMED (100 %)
16 μl
12 μl
Final volume
~4 ml
~8 ml
Note
For preparation of home-made acrylamide/bisacrylamide solutions use the formulas provided below:
%T = (AA(g) + BIS(g)) / mass of solution x 100 %C = BIS(g) / (AA(g) +BIS(g)) x 100
Procedure (for 0.75 mm minigel):
Pour 3.3 ml of the resolving gel solution between the glass plates with a pipette. Quickly but carefully apply 1.1 ml of the stacking gel solution. Avoid the formation of the bubbles.
Note
Because of the difference in densities of the stacking and resolving gel solutions, they do not mix with each other; a sharp interface is obtained immediately after applying the stacking gel solution.
Insert the comb. Ensure that no air bubbles are left in the gels. Allow the gels to polymerize for 1 hour at room temperature.
Note
For best results, keep the gel at 4°C overnight in a plastic bag with some electrophoresis running buffer (to avoid drying). Do not remove the combs.
Just prior to using the gel, remove the comb carefully. Place the gel into the electrophoresis tank, fill the tank (bottom and top reservoirs) with fresh 1X Tris-tricine-SDS buffer, making sure that the gel wells are covered with the buffer.
Load the samples.
Perform electrophoresis at 200 V for ~2 hours or until the dye front reaches the bottom of the gel.
Disassemble the gel sandwich and proceed with gel staining or Western blot procedures.
References
Shägger, H., and von Jagow, G., Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to100 kDa, Anal. Biochem, 166, 368-379, 1987.
Westermeier, R., Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations, 4th Edition, WILEY, 2004.
Kurien, B.T. and Scofield, R.H., Protein blotting: a review, J. Imm. Meth., 274, 1-15, 2003.
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