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Pre-Stained Protein Standards
(MultiMark®, SeeBlue®, SeeBlue® Plus2, BenchMark�, HiMark�)
TABLE OF CONTENTS
PRODUCT DESCRIPTION
Protein Origins
Molecular Weights
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
General Notes
Application Notes
Troubleshooting
ALTERNATE PRODUCTS & COMPATIBILITY
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBER
COMPONENTS
ASSOCIATED PRODUCTS
1
PRODUCT DESCRIPTION
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Protein Origins
Molecular Weights
Name Product Description
MultiMarkâ„? Multi- Contains 9 proteins that resolve into multi-colored bands in the range of 4 kDa to 250 kDa
Colored Standard (depending upon the buffer system). It is in a ready-to-use format. Suitable for Tris-
Glycine, Tricine, and NuPAGE Gels. No mixing, heating or reducing is required.
SeeBlue® Pre-Stained Contains 9 polypeptides that resolve into sharp, tight blue bands in the range of 4 kDa to
Standard 250 kDa (depending upon the buffer system). It is in a ready-to-use format. Suitable for
Tris-Glycine, Tricine, and NuPAGE Gels. No mixing heating or reducing is required.
SeeBlue® Plus2 Pre- Contains 10 proteins, eight dyed as in the SeeBlue® Standard, and two with contrasting
Stained Standard colors allowing easier band identification and sharp, distinct pre-stained bands. Proteins
resolve in the range of 4 kDa to 250 kDa (depending upon the buffer system). It is in a
ready-to-use format. Suitable for Tris-Glycine, Tricine, and NuPAGE Gels. No mixing,
heating or reducing is required.
BenchMark� Pre-Stained Contains 10 pre-stained recombinant protein bands in the range of 10 � 200 kDa. Proteins
are dyed blue with one orientation band (4th from top) dyed pink. It is in a ready-to-use
Protein Ladder
format. Suitable for Tris-Glycine type gels.
Note: Coupling of the chromophores to the proteins affects the apparent molecular weight
of the proteins in SDS-PAGE relative to unstained standards. Each pre-stained ladder is
calibrated against unstained standards and the apparent molecular weight is reported for
each lot of BenchMarkâ„? Pre-Stained Ladder. See certificate of analysis for lot specific
apparent molecular weights.
HiMarkâ„? Pre-Stained Contains 9 protein bands in the range of 30-460 kDa allowing for the determination of
High Molecular Weight apparent molecular weight of high molecular weight proteins on Tris-Acetate Gels with
(HMW) Protein Standard Tris-Acetate SDS buffer system. The marker is in a ready-to-use format. No mixing,
heating, or reducing isrequired.
Note: HiMarkâ„? Pre-Stained Standard can also be used with NuPAGE 4-12% Bis-Tris
Gels with MOPS Running Buffer and with Novex 4% Tris-Glycine Gels. However, to
obtain the best results with HMW proteins, always use NuPAGE Novex Tris-Acetate
Gels.
Protein Origins
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SeeBlue®, SeeBlue® Plus2, MultiMark�
Protein Origin
Myosin Rabbit muscle
B-galactosidase E. coli
Phosphorylase B Rabbit muscle
Bovine serum albumin Bovine serum
Glutamic dehydrogenase Bovine liver
Lactate dehydrogenase Porcine
Carbonic anhydrase Bovine
Trypsin inhibitor Soybean
Lysozyme Egg white
Aprotinin Bovine lung
Insulin B chain Bovine pancreas
Insulin A chain Bovine pancreas
Alcohol dehydrogenase Baker’s yeast
Myoglobin Horse heart
2
Protein Origins (BenchMarkâ„? Pre-Stained Standard)
The ladder consists of a series of affinity purified, recombinant proteins. The exact protein make up of each band is proprietary.
Approximate Molecular Weights for Gel Type
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MultiMarkâ„?
Tris Glycine Tricine NP MES NP MOPS NP TA
Myosin 250 kDa 208 kDa 185 kDa 188 kDa 209 kDa
Phosphorylase B 148 105 98 97 111
Glutamic Dehydrogenase 60 53 52 52 52
Carbonic Anhydrase 42 34 31 33 34
Myoglobin � Blue 30 23 19* 21* n/a
Myoglobin � Red 22 17 17* 19* n/a
Lysozyme 17 13 11 12 n/a
Aprotinin 6 7 6 n/a n/a
Insulin 4 4 3 n/a n/a
*NOTE - The 2 bands of Myoglobin red and blue are reversed in the NuPAGE Bis-Tris MES and MOPS buffers, compared to the
Tris-Glycine and Tricine systems.
SeeBlue®
Tris Glycine Tricine NP MES NP MOPS NP TA
Myosin 250 kDa 210 kDa 188 kDa 191 kDa 210 kDa
BSA 98 78 62 64 71
Glutamic Dehydrogenase 64 55 49 51 55
Alcohol Dehydrogenase 50 45 38 39 41
Carbonic Anhydrase 36 34 28 28 n/a
Myoglobin 30 23 18 19 n/a
Lysozyme 16 16 14 14 n/a
Aprotinin 6 7 6 n/a n/a
Insulin, B Chain 4 4 3 n/a n/a
SeeBlue® Plus2
Tris Glycine Tricine NP MES NP MOPS NP TA
Myosin 250 kDa 210 kDa 188 kDa 191 kDa 210 kDa
Phosphorylase B 148 105 98 97 111
BSA 98 78 62 64 71
Glutamic Dehydrogenase 64 55 49 51 55
Alcohol Dehydrogenase 50 45 38 39 41
Carbonic Anhydrase 36 34 28 28 n/a
Myoglobin Red 22 17 17 19 n/a
Lysozyme 16 16 14 14 n/a
Aprotinin 6 7 6 n/a n/a
Insulin, B Chain 4 4 3 n/a n/a
NOTE - Dyes used are proprietary. All the blue bands in either SeeBlue® or SeeBlue® Plus 2 have the same dye coupled to them.
In addition, all the blue bands in the BenchMarkâ„? Pre-stained Ladder use the same dye as well.
BenchMarkâ„? Pre-Stained Protein Ladder
Tris Glycine
3
1 ~190
2 ~120
3 ~85
4* ~60
5 ~50
6 ~40
7 ~25
8 ~20
9 ~15
10 ~10
NOTE � Apparent Molecular Weight is determined for each lot of BenchMark� Pre-Stained Protein Ladder. See Certificate of
Analysis for lot specific apparent MW’s. *Band 4 is the pink orientation band.
HiMarkâ„? Pre-Stained High Molecular Weight (HMW) Protein Standard
Band No. NuPAGE NuPAGE 4-12% 4% Tris-Glycine
Tris-Acetate Bis-Tris/MOPS
1 460 420 500
2 268 247 279
3 238 214 251
4* orientation band 171 160 164
5 117 107 121
6 71 64 n/a
7 55 51 n/a
8 41 39 n/a
9 31 28 n/a
SHIPPING CONDITIONS
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Name Primary Secondary
MultiMarkâ„? Multi-Colored Standard RT Wet Ice
SeeBlue® Pre-Stained Standard RT Wet Ice
SeeBlue® Plus2 Pre-Stained Standard RT Wet Ice
BenchMarkâ„? Pre-Stained Protein Ladder Dry Ice Wet Ice
HiMarkâ„? Pre-Stained High Molecular Weight (HMW) Dry Ice Dry Ice
Protein Standard
STORAGE CONDITIONS
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Name Storage
MultiMark� Multi-Colored Standard -20°C
SeeBlue® Pre-Stained Standard +4°C (can also be stored at -20°C)
SeeBlue® Plus2 Pre-Stained Standard +4°C (can also be stored at -20°C)
BenchMark� Pre-Stained Protein Ladder -20°C (Avoid repeated freezing and thawing)
HiMark� Pre-Stained High Molecular -20°C
Weight (HMW) Protein Standard
STABILITY
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Name Stability
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MultiMarkâ„? Multi-Colored Standard Guaranteed stable for 3 months when properly
stored.
SeeBlue® Pre-Stained Standard Guaranteed stable for 4 months when properly
stored.
SeeBlue® Plus2 Pre-Stained Standard Guaranteed stable for 4 months when properly
stored.
BenchMarkâ„? Pre-Stained Protein Ladder Guaranteed stable for 1 year when properly
stored.
[Stable 2.5 Years at -20°C, 1 month only at
4°C, 1 week only at RT, 2 days only at 37°C.
Stable for 50 freeze/thaws.]
HiMark� Pre-Stained High Molecular Stable for 4 months at -20°C
Weight (HMW) Protein Standard
QC SPECIFICATIONS
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MultiMarkâ„? Multi-Colored Standard
Gel Testing: 10% Tris-Glycine, 10�20% Tricine, and 10% Bis-Tris.
Nine bands must be present on the 10�20% TR and the 10% BT.
Gel test performance must be comparable to 2 previously released lots.
Band intensity must not differ more than 20% from 2 previously released lots.
Bands must migrate to within one half band width of the reference lots.
Contaminant bands must be less than 20% of the intensity of the major bands.
SeeBlue® Pre-Stained Standard
Gel Testing: 8%Tris-Glycine, 12%Tris-Glycine, 10�20%Tricine, and 10%Bis-Tris.
Nine bands must be present on the 10�20%TR and the 10%BT.
Gel test performance must be comparable to 2 previously released lots.
Band intensity must not differ more than 20% from 2 previously released lots.
Bands must migrate to within one half band width of the reference lots.
Contaminant bands must be less than 20% of the intensity of the major bands, except for those associated with insulin.
For insulin A, contaminant band must be less than 25% of the intensity of the major bands in previously released lots.
SeeBlue® Plus2 Pre-Stained Standard
Gel Testing: 8% Tris-Glycine, 12% Tris-Glycine, 10�20% Tricine and 10% Bis-Tris. *Ten bands must be present on the
10�20% TR and the 10% BT gels.
Gel test performance must be comparable to 2 previously released lots.
Band Intensity must not differ more than 20% from 2 previously released lots.
Bands must migrate to within one half band width of those in the reference lots.
Contaminant bands must be less than 20% of the intensity of the major bands, except those associated with Insulin. For
Insulin A, contaminant band must be less than 25% of the intensity of the major bands in previously released lots.
BenchMarkâ„? Pre-Stained Protein Ladder
Gel Testing: Ten major bands must be present on a 4�20% Tris−Glycine gel.
Band intensity must not differ more than 20% from a previously released lot.
Migration: Apparent molecular weights of the ladder are determined on a 4�20% Tris−Glycine gel by comparing the
mobility of the BenchMarkâ„? Pre-stained Protein Ladder to the unstained BenchMarkâ„? Protein Ladder. The apparent
MW for each lot is recorded on the certificate of analysis for that lot.
HiMarkâ„? Pre-Stained High Molecular Weight (HMW) Protein Standard
Gel Testing: Nine bands must be present on both a 7% and 3~8% Tris-Acetate gels. Performance must be comparable to
two previously released lots.
Migration: Band migration must be straight and sharp, and within 1/2 bandwith of two previously released lots.
Intensity: Band intensity must not differ more than 20% from two previously released lots.
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PROTOCOL AND APPLICATION NOTES
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General Notes
Application Notes
Troubleshooting
General Notes
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These protein standards are for SDS-PAGE and should not to be used for native electrophoresis. These markers are
denatured and have SDS in the storage buffer.
These standards are ready to load. There is no need to heat or add reducing agents. Do not boil - boiling may cause band
degradation. Proteins are alkylated to make re-oxidation impossible and to make these protein markers ready-to-use.
Pre-stained standards are ideal for monitoring an electrophoresis run, estimating the efficiency of transfer onto a
membrane, and determining the approximate molecular weight of proteins. For accurate molecular weight estimation of
proteins, use unstained protein ladders.
The molecular weight values for the proteins in a pre-stained protein standard will be different in different gel types. The
molecular weight values that are stated in conjunction with our standards are given as "apparent" molecular weights. The
differences between the apparent molecular weights and the published molecular weights are a result of the covalent
attachment of dye to the proteins used in the marker. The bound dye molecules can carry a charge. Of course, this charge
changes the ability of the SDS to bind to the protein in addition to contributing directly to the protein's charge. The result
is a protein with an altered charge and consequent change in mobility within the gel. This explains why the proteins in
pre-stained markers run differently from their unstained counterparts.
Further difference observed between the same marker in differing gel types (Tris-Glycine vs. NuPAGE for example) is
possibly because of the different pHs of the gel types. At higher pH values (TG gels), charges are more likely to be
protonated. Meanwhile, at the lower more neutral pH of a NuPAGE gel, the charges are more skewed towards
deprotonation, giving the same stained proteins a more negative charge overall. In an SDS PAGE system, more negative
charge means more mobility. This is why the same pre-stained protein will be "larger" on a Tris-Glycine gel than a
NuPAGE gel. In the NuPAGE gel, the lower (~ neutral) pH causes more overall negative charge, making the apparent
molecular weight to be much smaller. This effect generally increases in magnitude with the size of the protein and is
greatest with myosin due to the increase in dye binding sites. Please see apparent molecular weights of our SeeBlue®,
SeeBlue® Plus2, and MultiMark� in different buffers and gels in the respective manuals.
Application Notes
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Quantitation/Determining Protein Concentration
These pre-stained markers are not designed for the quantitation and it is not recommended that these markers be used in the
determination of protein concentration. Estimations of the protein concentration for SeeBlue®, SeeBlue® Plus2, and
BenchMarkâ„? Pre-Stained Standards are given below but these values are an approximation and should not be used to quantify
samples.
SeeBlue®/SeeBlue® Plus2
Band per 10 µl
Myosin 2.20 µg
Phosphorylase-b* *Only in SeeBlue® Plus2. No estimation available
BSA 0.75 µg
GDH 1.25 µg
Alcohol Dehydrogenase 0.80 µg
Carbonic Anhydrase 0.90 µg
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Myoglobin (blue)* 1.50 µg *Myoglobin (red) in SeeBlue® Plus2. No estimation available.
Lysozyme 2.50 µg
Aprotinin 1.80 µg
Insulin 2.50 µg
BenchMark� Pre-Stained - Protein/Band is ~0.1 µg/µl
BenchMarkâ„? Pre-Stained Standard and NuPAGE Gels: Since the migration of a pre-stained standard is affected by the
pH of the gel and buffer system, the apparent molecular weights of the BenchMarkâ„? Pre-Stained Standard will be
different in a NuPAGE gel than in a Tris-Glycine type gel. One can calibrate how a given lot of BenchMarkâ„? Pre-
Stained Standard runs on a NuPAGE gel by comparing the migration to an unstained standard.
Western blotting with HiMarkâ„? Pre-Stained HMW Protein Standard
Nitrocellulose Membranes - Perform transfer with 1X transfer buffer with 10% methanolusing the recommended
transfer conditions based on the gel type. Use 1:1000 NuPAGE Antioxidant for efficient transfer of reduced proteins.
PVDF Membranes - Perform transfer with 1X transfer buffer with 20% methanol using the recommended transfer
conditions based on the gel type. Use 1:1000 NuPAGE Antioxidant for efficient transfer of reduced proteins.
For a detailed blotting protocol for the efficient transfer of large proteins, refer to the NuPAGE Large Protein
Analysis System manual.
Troubleshooting
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Missing bands
Check % gel that is being used. Depending on gel type and/or percentage, you may not see all the bands. For example,
one would not see the smallest bands of the standard on a very low % gel. A high % gel may not resolve the higher MW
bands.
Check age of ladder � expired lots may see faded or missing bands.
Check storage of ladder � adverse storage conditions will affect the stability of the ladder.
Boiling of ladder may contribute to degradation/missing bands (BenchMarkâ„? Pre-Stained Standard pink orientation band
is the first to be lost upon boiling).
Smeary bands
Don’t load too much protein. See recommended load volumes in the manual.
Bands will not be as well resolved on low % gels. Try using gel % greater than 8%.
Extra Bands
Too much protein was loaded per lane. This is especially a problem with silver stained gels.
Cross contamination in the lane from adjacent samples.
Marker has wrong MW on gel/blot
Pre-Stained Standards show an apparent MW that is affected by pH of the gels and buffers used. Please see discussion of
molecular weight values above in the ‘General Notes� Section.
Don’t use these markers on a native gel. These markers are only for SDS-PAGE.
For more information on factors affecting protein migration:
Tung, Jwu-Sheng and Knight, C.A. (1972) Analytical Biochemistry 48, 153-163
Matagne,A., Joris,B., Frere, J. (1991) Biochem J. 280, 553-556
Cross Reactivity on Western Blots
See protein origins above in ‘Components� section.
Poor Transfer
Increase voltage, current or length of time for transfer.
For transfer to PVDF, omit the SDS from the transfer buffer. SDS will cause the proteins to bind less efficiently to
membranes because it disrupts the hydrophobic interaction between the membrane and the protein. If SDS is present in
transfer buffer (i.e. to facilitate transfer of large proteins), make sure that there is no more than 0.02% SDS present in
buffer.
Methanol helps to enhance the hydrophobic “stick� of the proteins to the membrane. Too much methanol however, can
be a problem as the proteins can become fixed in the gel. The methanol concentration in a western blot should be
between 10 � 20%
Marker goes through membrane during transfer
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Decrease voltage, transfer time.
Ensure proper SDS, methanol concentration. Too much SDS can prevent binding to membrane. Alcohol enhances
hydrophobic binding to membrane; not enough alcohol may prevent binding.
Check pore size of membrane and size of proteins of interest. Proteins smaller than 10kDa will more easily transfer
through a 0.45um pore size membrane. If smaller proteins are the target, 0.2um pore size will better capture those
proteins smaller than 10kDa.
Bands fade during Western
It is common for any pre-stained protein standard to fade during the blocking step of a western blot. After transfer, it is a
good idea to circle the pre-stained bands with a pencil on the membrane, so band positions can be identified after blocking
and processing.
PRODUCT DOCUMENTATION
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Brochures Citations Cell lines
COA FAQ Licensing
Manuals MSDS Newsletters
PRODUCT NAME AND CATALOG NUMBERS
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Name Size Catalog Number
500 µl (50 applications of 10 µl each)
MultiMarkâ„? Multi-Colored Standard LC5725
500 µl (50 applications of 10 µl each)
SeeBlue® Pre-Stained Standard LC5625
500 µl (50 applications of 10 µl each)
SeeBlue® Plus2 Pre-Stained Standard LC5925
2 x 250 µl (50 applications of 10 µl each)
BenchMarkâ„? Pre-Stained Protein Ladder 10748010
HiMarkâ„? Pre-Stained High Molecular 250 ul (25 applications of 10 ul each) LC5699
Weight (HMW) Protein Standard
COMPONENTS
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Name Components
500 µl supplied in loading buffer containing Tris-HCl, Formamide,
MultiMarkâ„? Multi-Colored
Standard SDS, and Phenol Red. The SDS concentration is 1%.
500 µl supplied in loading buffer containing Tris-HCl, Formamide,
SeeBlue® Pre-Stained Standard
SDS, and Phenol Red.
500 µl supplied in loading buffer containing Tris-HCl, Formamide,
SeeBlue® Plus2 Pre-Stained
Standard SDS, and Phenol Red.
Two vials of 250 µl each are provided in loading buffer consisting of
BenchMarkâ„? Pre-Stained Protein
Ladder 50 mM Tris-HCl, pH 6.8; 5 mM EDTA; 10 mM DTT; 1% (w/v)
SDS; 10% (w/v) glycerol
250 µl of HiMark� Pre-Stained HMW Protein Standard supplied in
HiMarkâ„? Pre-Stained High
Molecular Weight (HMW) storage buffer consisting of Tris-HCl, Formamide, SDS, & Phenol
Protein Standard Red
ASSOCIATED PRODUCTS
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Novex Tris-Glycine Gels
NuPAGE Bis-Tris Gels
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NuPAGE Tris-Acetate Gels
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