Protein Standards and Ladders Support — Getting Started

Except for our NativeMark™ Unstained Protein Standard (designed for native electrophoresis), all of the other unstained and prestained standards we offer (Novex™ Sharp, SeeBlue™, SeeBlue™ Plus2, BenchMark™, HiMark™) have been pre-reduced (by a proprietary method). Hence, you do not need to add reducing agent.

Our protein standards are ready to load. We do not recommend heating them as this may cause protein degradation.

Please find this information in the respective manuals for the individual protein standards.

We recommend using unstained protein ladders for molecular weight estimation applications as prestained ladders have a dye that is covalently bound to each protein that will result in the ladder migrating differently in different buffer systems (i.e., different gels). As a result, using a prestained ladder for molecular weight estimation will only give the apparent molecular weight of the protein. Prestained ladders may be used for molecular weight approximation, confirming gel migration and estimating blotting efficiency but for accurate molecular weight estimation, an unstained ladder should be used.

Our protein standards are not designed for protein quantitation.

Zymogram gels are essentially Tris-Glycine gels containing the substrate. Protein standards run based solely on the percentage of acrylamide and hence should run the same in both kinds of gels. It is quite possible though that if the standard is prestained, the proteins will appear a different color because of the staining (or pre-staining) of the Zymogram gels.

Yes, we recommend the Novex™ Sharp Prestained Protein Standard (Cat. No. LC5800). It consists of 12 prestained protein bands in the range of 3.5 kDa to 260 kDa. Each band is stained in a distinct color for easy identification, and the bands are extremely tight and sharp for the most accurate molecular weight estimation. The marker is ready-to-use under denaturing and reducing conditions and can be used on NuPAGE™ Bis-Tris, Tris-Glycine, and Tricine gels.

Our protein standards are not designed for protein quantitation.

We recommend storing these ladders at –20 degrees C where they are stable for a year. They are stable for up to 3 months at 4 degrees C.

The PageRuler™ Plus Prestained Protein Ladder is a mixture of nine (9) blue-, orange- and green-stained proteins (10 to 250 kDa) for use in protein electrophoresis (SDS-PAGE) and western blotting. Two orange reference bands at ~70 kDa and 25 kDa and one green reference band at 10 kDa highlight the blue-stained protein ladder.

These ladders contain recombinant prokaryotic proteins and do not contain any animal-derived proteins.

The PageRuler™ Prestained Protein Ladder is a mixture of ten (10) blue-, orange- and green-stained proteins (10 to 180 kDa) for use in protein electrophoresis (SDS-PAGE) and western blotting. The ladder contains one orange reference band at 70 kDa and one green band at 10 kDa.

We recommend storing the Spectra™ Protein Ladders at –20 degrees C where they are stable for a year. The Spectra™ Multicolor Broad Range Protein Ladder and Spectra™ Multicolor High Range Protein Ladder are stable for up to 3 months at 4 degrees C. The Spectra™ Multicolor Low Range Protein Ladder is stable for up to 2 months at 4 degrees C.

The Spectra™ Multicolor Broad Range Protein Ladder is a mixture of 10 recombinant prestained proteins ranging from 10 kDa to 260 kDa, for use in gel electrophoresis and western blotting. Four different chromophores are bound to the proteins, producing a brightly colored ladder.

The Spectra™ Multicolor High Range Protein Ladder is a mixture of eight (8) blue-, green-and orange-stained proteins (40 to 300 kDa) for use as a high-MW standard in gel electrophoresis and western blotting. Three different chromophores are bound to the proteins, producing a brightly colored ladder.

The Spectra™ Multicolor Low Range Protein Ladder is a prestained mixture of six recombinant proteins (1.7 to 40 kDa) for use in gel electrophoresis and western blotting. Three different chromophores are bound to the proteins, producing a brightly colored ladder.

The Spectra™ Protein Ladders contain recombinant prokaryotic proteins and do not contain any animal-derived proteins.

We recommend storing the HiMark™ Prestained Protein Standard at –20 degrees C. The ladder is stable for 4 months when properly stored.

We recommend using the HiMark™ Prestained Protein Standard, Cat. No. LC5699 for determination of apparent molecular weight of high molecular weight proteins on NuPAGE™ Tris-Acetate gels with Tris-Acetate SDS buffer system. For accurate estimation of molecular weight of high molecular weight proteins on NuPAGE™ Tris-Acetate gels with Tris-Acetate SDS buffer system, we recommend using the HiMark™ Unstained Protein Standard, Cat. No. LC5688.

We recommend storing the Novex™ Sharp Prestained Protein Standard at –20 degrees C. It is stable for 12 months when properly stored.

The proteins in the Novex™ Sharp Prestained Protein Standard are synthetic fusion proteins and their origins are proprietary. The 10, 20, 60 and 80 kDa proteins in the Novex™ Sharp Prestained Standard have C-terminal 6xHis tags and hence are shown to cross react with the anti-His C-term antibody.

The Novex™ Sharp Prestained Standard has not been tested on zymogram gels. We recommend using the SeeBlue™ Plus2 Prestained Standard (at 2–3X concentration) for those gels.

The Novex™ Sharp Prestained Protein Standard is suitable for NuPAGE™ Bis-Tris, Tris-Glycine and Tricine gels. This standard has not been tested on NuPAGE™ Tris-Acetate gels. The dyes that are covalently bound to the proteins in the Novex™ Sharp Prestained Protein Standard were selected to minimize any effect they may have on migration of the ladder in different gel chemistries. As a result, the apparent molecular weights of the proteins are the same in NuPAGE™ Bis-Tris, Tris-Glycine and Tricine gels. Below is a figure showing the apparent molecular weight of each band. Note that the 3.5 kDa band is visible only on NuPAGE™ Bis-Tris gels run using MES SDS Running buffer or Tricine gels.

The SeeBlue™ Prestained Standard has a blue cap whereas the SeeBlue™ Plus 2 Prestained Standard has a reddish orange cap.

Here is a table showing the origins of the proteins in the SeeBlue™ Prestained Standard and SeeBlue™ Plus 2 Prestained Standard:

The dyes used are proprietary. All the blue bands in the SeeBlue™ Prestained Standard (9 blue bands) and SeeBlue™ Plus 2 Prestained Standard (8 blue bands) have the same dye coupled to them. In addition, the SeeBlue™ Plus 2 Prestained Standard has two colored bands.

We recommend storing the SeeBlue™ Prestained Standard and SeeBlue™ Plus 2 Prestained Standard at 4 degrees C. They are stable for 4 months when properly stored.

The BenchMark™ Prestained Protein Ladder contains a series of affinity purified, recombinant proteins. The exact make-up and origin of each band is proprietary.

We recommend storing the BenchMark™ Prestained Protein Ladder at –20 degrees C. It is stable for 1 year when properly stored.

The BenchMark™ Prestained Protein Ladder is designed for use with Tris-Glycine gels alone, when run with Tris-Glycine SDS Running buffer.

The dyes used are proprietary. All the blue bands in the BenchMark™ Prestained Protein Ladder have the same dye coupled to them.

We recommend storing the Thermo Scientific™ Prestained Protein MW Marker at –20 degrees C where it is stable for a year.

The Thermo Scientific™ Prestained Protein MW Marker is a mixture of six blue-stained natural proteins ranging from 20 kDa to 120 kDa.

The iBright Prestained Protein Ladder has been optimized and designed for use with western blotting as it provides 2 control bands (unstained ladder bands) that are detected using the same antibody conjugate and protocol. The 2 unstained control bands contain IgG binding sites that can be visualized simultaneously with your target without any additional steps in the protocol. The 10 stained bands will appear during electrophoresis and transfer. These 10 bands will also appear in fluorescent imaging in the NIR channels. The 2 control bands will appear with chemiluminescent or fluorescent detection similar to your target.

If performing detection on a mini gel, the recommended loading volume is 1-3 µL. If performing visualization on a midi gel, 2-4 µL is recommended, and for detection, 2-3 µL is recommended. Optimal loading volume should be determined empirically.

The 2 unstained bands in the iBright Prestained Ladder contain an IgG binding site, allowing direct visualization with the same antibody-conjugate reagents used to detect the target protein. The proteins in the standard will not bind to IgM antibodies.

Most of the common gel running buffers are composed of Tris-glycine or Tris-tricine. Tris-glycine buffer systems are useful for separation of proteins over a wide range of molecular weights (5-300 kDa) and are compatible with denaturing or non-denaturing conditions. Tris-tricine buffer is generally recommended for the electrophoresis of low molecular weight proteins and peptides (<10 kDa) that need to be reduced and denatured prior to loading. Tris-acetate buffer system is used for separation of larger proteins (>200 kDa).

We recommend storing the PageRuler™ Unstained Protein Ladders at –20 degrees C where they are stable for a year.

The PageRuler™ Unstained Low Range Protein Ladder is a mixture of seven recombinant proteins ranging from 5 kDa to 100 kDa and a synthetic peptide at 3.4 kDa. For easy reference, the 25 kDa protein band is of greater intensity.

The PageRuler™ Unstained Protein Ladder is a mixture of 14 recombinant, highly purified proteins ranging from 10 kDa to 200 kDa. For easy reference, the 50 kDa protein band has a greater intensity than the other proteins in the ladder.

The proteins in the different PageRuler™ Unstained Protein Ladders (except for the 3.4 and 5 kDa peptides in the PageRuler™ Unstained Low Range Protein Ladder and the PageRuler™ Unstained Protein Ladder, and the 5 kDa peptide in the PageRuler™ Unstained Broad Range Protein Ladder) contain an integral Strep-tag™ II Sequence and may be detected on western blots using Strep-Tactin™ Conjugates.

The PageRuler™ Unstained Protein Ladder, PageRuler™ Unstained Broad Range Protein Ladder, and PageRuler™ Unstained Low Range Protein Ladder contain recombinant prokaryotic proteins and do not contain any animal-derived proteins.

The PageRuler™ Unstained Broad RangeProtein Ladder is a mixture of eleven recombinant, highly purified proteins ranging from 5 kDa to 250 kDa. For easy reference, the 100 kDa, 50 kDa and 20 kDa protein bands have greater intensity than the other proteins in the ladder.

We recommend storing the HiMark™ Unstained Protein Standard at –20 degrees C in aliquots to avoid repeated freeze thawing. The ladder is stable for 6 months when properly stored.

The HiMark™™ Unstained Protein Standard allows the accurate estimation of molecular weight of high molecular weight proteins on NuPAGE™ Tris-Acetate Gels with Tris-Acetate SDS buffer system. Here is a table showing the molecular weights of the proteins in the HiMark™ Unstained Protein Standard:

For accurate estimation of molecular weight of high molecular weight proteins on NuPAGE™ Tris-Acetate gels with Tris-Acetate SDS buffer system, we recommend using the HiMark™ Unstained Protein Standard, Cat. No. LC5688.

We recommend storing the Novex™ Sharp Unstained Protein Standard at –20 degrees C. It is stable for 12 months when properly stored.

The proteins in the Novex™ Sharp Unstained Protein Standard are synthetic fusion proteins and their origins are proprietary. The 10, 20, 60 and 80 kDa proteins in the Novex™ Sharp Unstained Protein Standard have C-terminal 6xHis tags and hence are shown to cross react with the anti-His C-term antibody.

The Novex™ Sharp Unstained Protein Standard has not been tested on zymogram gels. We recommend using the SeeBlue™ Plus2 standard (at 2–3X concentration) for those gels.

Below is a figure showing the molecular weights of the proteins in the Novex™ Sharp Unstained Protein Standard. These molecular weights apply to NuPAGE™ Bis-Tris, Tris-Glycine and Tricine gels. This standard has not been tested on NuPAGE™ Tris-Acetate gels. Note that the 3.5 kDa band is visible only on NuPAGE™ Bis-Tris gels run using 1X MES SDS Running buffer or Tricine gels. This band is also not visible when stained with SYPRO Ruby’s microwave staining protocol.

We recommend storing the BenchMark™ Unstained Protein Ladder at –20 degrees C. It may also be stored at 4 degrees C. The ladder is stable for six months when properly stored.

The BenchMark™ Unstained Protein Ladder contain a series of affinity purified, recombinant proteins. The exact make-up and origin of each band is proprietary.

The BenchMark™ Unstained Protein Ladder is suitable for NuPAGE™, Tris-Glycine and Tricine gels. Here is a table showing the molecular weights of the proteins in the ladder:

*20 kDa and 50 kDa bands are darker in intensity and serve as orientation bands.

Here is a table showing the isoelectric points for the proteins in the BenchMark™ Unstained Protein Ladder:

The BenchMark™ Unstained Protein Ladder can be used for SDS-PAGE with NuPAGE™ gels, Tris Glycine gels or Tricine gels.

We recommend storing the Mark12™ Standard at 4 degrees C and it is stable for 6 months when properly stored.

The Mark12™ Unstained Standard is suitable for NuPAGE™, Tris-Glycine and Tricine gels. Here is a table showing the molecular weights and origins of the proteins in the Mark12™ Unstained Standard:

Note: The 3.5 kDa and 2.5 kDa bands are visible only on NuPAGE™ Bis-Tris gels run using 1X MES SDS Running buffer.

We recommend storing the Thermo Scientific™ Unstained Protein MW Marker at –20 degrees C where it is stable for a year.

The Thermo Scientific™ Unstained Protein MW Marker is a mixture of seven natural proteins ranging from 14.4 kDa to 116 kDa.

We recommend storing the IEF Marker 3–10 at –20 degrees C and it is stable for 1 year when properly stored.

The IEF Marker 3–10 is a ready-to-use, unstained protein marker especially developed for IEF applications. It contains 13 purified proteins ranging in pI from 3–10. It works well on horizontal and vertical one-dimensional IEF gels and can be used for both native and denaturing conditions (e.g., 8 M urea, to determine the isoelectric point (pI) of unknown protein samples. Here is a table showing the pIs and origins of the proteins in the IEF Marker, 3–10:

*Temperature = 5 degrees C for values given. pI is temperature dependent, i.e. decreases as temperature increases (approximately –0.005 to –0.03 pH units per degree C)

c = cathodic; m = middle; a = anodic

The IEF Marker, 3–10 is not designed for use with IPG strips.

The exact amount of each protein in the IEF Marker, 3–10 is not known. We do not recommend using this marker for quantitation purpose.

We recommend using the NativeMark™ Unstained Protein Standard, Cat. No. LC0725 for native gel electrophoresis with Tris-Glycine, NuPAGE™ Tris-Acetate or NativePAGE™ gels.

We recommend storing the NativeMark™ Unstained Protein Standard at –20 degrees C in aliquots to avoid repeated freezing and thawing. After a vial is thawed for use, we recommend storing it at 4 degrees C for up to one month. It is stable for 6 months when stored at –20 degrees C.

The NativeMark™ Unstained Protein Standard allows the estimation of molecular weight of proteins when performing native electrophoresis. It is suitable for use with NativePAGE™ Bis-Tris gels, or gradient Tris-Glycine and Tris-Acetate gels run using Tris-Glycine Native Running buffer. Here is a table showing the molecular weights of the proteins in the NativeMark™ Unstained Protein Standard:

*Note: The B-phycoerythrin band may appear pink on the gel as the protein has an intrinsic pink color and autofluoresces.

The IgM Hexamer and IgM Pentamer protein bands in the NativeMark™ protein standard are bovine in origin.

The NativeMark™ Unstained Protein Standard is designed for use with native gels such as NativePAGE™ Bis-Tris gels, or gradient Tris-Glycine and Tris-Acetate gels run using Tris-Glycine Native Running buffer. Using this standard with denaturing gels will result in inaccurate molecular weight estimation.

We recommend storing the BenchMark™ His-tagged Protein Standard at –20 degrees C in aliquots to avoid repeated freezing and thawing. It is stable for 6 months when properly stored.

The BenchMark™ His-tagged Protein Standard is designed for easy visualization of protein molecular weight ranges of His-tagged fusion proteins after electrophoresis or western blotting. Each protein in the standard contains a 6xHis-tag enabling detection with InVision™ His-tag In-gel Stain, Cat. No. LC6030 or with Anti-His(C-term) Antibody, Cat. No. R930-25. The standard can also be detected with Coomassie™ staining. It is suitable for use with NuPAGE™ or Tris-Glycine gels. Here is a table showing the apparent molecular weights of the proteins in the BenchMark™ His-tagged Protein Standard:

The BenchMark™ His-tagged Protein Standard cannot be detected using anti-HisG antibody. It can be detected using Anti-His(C-term) Antibody, Cat. No. R930-25.

The identity of each protein in the BenchMark™ His-tagged Protein Standard is proprietary.

The concentration of each protein in the standard is proprietary. We do not recommend using this standard for quantitation purpose.

We recommend storing the BenchMark™ Fluorescent Protein Standard in the dark at –20 degrees C and in aliquots to avoid repeated freezing and thawing. It is stable for 6 months when properly stored.

The BenchMark™ Fluorescent Protein Standard is designed for visualization of molecular weight ranges of proteins labeled with Lumio™ Green Detection kit (Cat. No. LC6090) or other fluorescent-conjugated proteins. The standard consists of a set of wide molecular weight range proteins conjugated to a fluorescent dye, Alexa Fluor™ 488. The standard can also be detected with Coomassie staining. It is suitable for use with NuPAGE™ or Tris-Glycine gels. Here is a table showing the apparent molecular weights of the proteins in the BenchMark™ Fluorescent Protein Standard in the different gels:

The maximum excitation of the dye is at 493 nm and the maximum emission is at 516 nm.

The concentration of each protein in the standard is proprietary. We do not recommend using the standard for quantitation purpose.

The identity of each protein band in the BenchMark™ Fluorescent Protein Standard is proprietary.

We recommend storing the PageRuler™ Prestained NIR Protein Ladder at –20 degrees C where it is stable for a year.

The PageRuler™ Prestained NIR Protein Ladder is a mixture of 10 proteins (11 kDa to 250 kDa) that are blue-stained and fluor-labeled for near-IR (NIR) fluorescent visualization and protein sizing following electrophoresis. The 55kDa band is of greater intensity and serves as a reference band.

The PageRuler™ Prestained NIR Protein Ladder contains recombinant prokaryotic proteins and does not contain any animal-derived proteins.

We recommend storing the E-PAGE™ SeeBlue™ Prestained at 4 degrees C and it is stable for 4 months when properly stored.

The E-PAGE™ SeeBlue™ Prestained Standard is designed for use with E-PAGE™ pre-cast gels, to visualize protein molecular weight ranges during electrophoresis and evaluate western transfer efficiency. The apparent molecular weights of the proteins in the E-PAGE™ SeeBlue™ Prestained Standard are shown below:

Each of the six proteins is present at approximately 0.5 µg/µL concentration.

For long-term storage, store the stock tube at either –20 degrees C or –80 degrees C. For short-term storage, store at 2–6 degrees C. The stock can be subjected to repeated freezing and thawing without any deleterious effects to the molecular weights of the proteins or their migration during electrophoresis.

All proteins are single chain, monomeric proteins (no subunits). The phosphoproteins are both serine-phosphates. Ovalbumin has 2 phosphorylated residues and β-casein has 5 phosphorylated residues. We do not determine whether or not all the phosphates are present at all times for each lot and can only assume that a majority of each protein has all sites phosphorylated. Any protein extracted from a natural source may always have the potential for some dephosphorylation during extraction/purification or by natural cellular activity. For a more definitive phosphorylation number comparison to staining intensity, a synthesized peptide of known number of phosphates must be used. Please see table below for details:

Each of the eight proteins is present at approximately 0.5 µg/µL concentration.

For long-term storage, store the stock tube at either –20 degrees C or –80 degrees C. For short-term storage, store at 2–6 degrees C. The stock can be subjected to repeated freezing and thawing without any deleterious effects to the molecular weights of the proteins or their migration during electrophoresis.

All proteins are single chain, monomeric proteins (no subunits). Please see table below for details:

Each of the eleven proteins is present at approximately 60 µg/mL concentration.

For long-term storage, store the stock tube at either –20 degrees C or –80 degrees C. For short-term storage, store at 2–6 degrees C. The stock can be subjected to repeated freezing and thawing without any deleterious effects to the molecular weights of the proteins or their migration during electrophoresis.

What are the characteristics of the proteins included in the Protein Molecular Weight Standards (broad range)?

We recommend storing the MagicMark™ XP Western Protein Standard –20 degrees C in aliquots to avoid repeated freezing and thawing. It is stable for 4 months when properly stored.

The MagicMark™ XP Western Protein Standard is designed for molecular weight estimation of proteins directly on western blots. Each protein in the standard contains an IgG binding site, allowing direct visualization with the same antibody-conjugate reagents used to detect the target protein on blots. Colorimetric, chemiluminescent, or fluorescent detection methods can be used for analysis. MagicMark™ XP Western Protein Standard is compatible with NuPAGE™ and Tris-Glycine gels. Here is a table showing the molecular weights of the proteins in the MagicMark™ XP Western Protein Standard:

The MagicMark™ XP protein bands can be visualized with the same antibody-conjugate reagents used to detect the target protein, using either colorimetric or chemiluminescent methods. MagicMark™ XP standard can be used for molecular weight estimation of proteins. Each protein in the MagicMark™ XP Western Protein Standard contains an IgG binding site. The proteins in the standard will not bind to IgM antibodies.

Each protein in the MagicMark™ XP Western Protein Standard contains an IgG binding site, allowing direct visualization with the same antibody-conjugate reagents used to detect the target protein. The proteins in the standard will not bind to IgM antibodies.

You can combine 5 μL MagicMark™ XP Standard with 5 μL Novex™ Sharp Prestained Standard or 10 μL MagicMark™ XP Standard with 5 μL SeeBlue™ Prestained Standard in the same run using the same lane. The colored bands from the prestained standard can be used to confirm gel run and transfer. Following immunodetection, sharp MagicMark™ XP bands will develop directly on the western blot.

Here is a table showing the relative affinities of the MagicMark™ XP Western Protein Standard to IgG antibodies from different species:

Enzyme-conjugated primary antibodies may not bind efficiently with the proteins in the MagicMark™ XP Western Protein Standard. We recommend using unconjugated primary antibody, followed by the addition of enzyme-conjugated secondary antibody.

Note: The Anti-myc-AP/HRP and Anti-V5-AP/HRP antibodies do not bind to MagicMark™ XP proteins.

The exact protein amount in each band of the standard is proprietary. We do not recommend using this standard for quantitation purpose.

We recommend storing these ladders at –20 degrees C where they are stable for a year. They are stable for up to one month at room temperature and for up to three months at 4 degrees C.

The SuperSignal™ Molecular Weight Protein Ladder is a mixture of eight recombinant proteins ranging from 20 kDa to 150 kDa. Each protein in the mixture contains an IgG-binding site and is proportioned to yield comparable gel electrophoresis and western blotting band intensities. The ladder contains a pink tracking dye in the buffer for monitoring electrophoresis and transfer. The ladder can be used with chemiluminescent, fluorescent, chromogenic and other detection types.

The SuperSignal™ Enhanced Molecular Weight Protein Ladder is a mixture of eight recombinant proteins ranging from 20 kDa to 150 kDa. Each protein in the mixture contains an IgG-binding site and is proportioned to yield comparable gel electrophoresis and western blotting band intensities. The ladder contains a pink tracking dye in the buffer for monitoring electrophoresis and transfer. The ladder can be used with chemiluminescent, fluorescent, chromogenic and other detection types. This ladder is formulated specifically for antibody species that do not bind well to other antibody-based ladders, such as mouse monoclonal antibodies.

We would recommend using the SuperSignal™ Molecular Weight Protein Ladder for most applications. Use the SuperSignal™ Enhanced Molecular Weight Protein Ladder when low concentrations of antibody are used or when mouse primary antibody is used.

We recommend storing the E-PAGE™ MagicMark™ Unstained Protein Standard at –20 degrees C in aliquots to avoid repeated freezing and thawing. It is stable for 6 months when properly stored.

The E-PAGE™ MagicMark™ Unstained Protein Standard is designed for use with E-PAGE™ pre-cast gels, for estimating the molecular weight of proteins after staining or western blotting. Each protein in the standard contains an IgG binding site. The standard can also be visualized with SimplyBlue™ SafeStain, silver stain, or fluorescent stains. The molecular weights of the proteins in the E-PAGE™ MagicMark™ Unstained Protein Standard are shown below:

Here is a table showing the relative affinities of the E-PAGE™ MagicMark™ Unstained Protein Standard to IgG antibodies from different species: