Thermo Scientific™ Spectra™ Multicolor Low Range Protein Ladder

Catalog No.	Quantity
PI26628	250 μL

Catalog No. PI26628 Supplier Thermo Scientific™ Supplier No. 26628

Description

A mixture of six proteins and synthetic peptides (1.7 to 40kDa) for use as size standards for peptides and small proteins in gel electrophoresis and Western blotting.

Thermo Scientific™ Spectra Multicolor Low Range Protein Ladder is a mixture of six (6) proteins and synthetic peptides (1.7 to 40kDa) for use as size standards for peptides and small proteins in gel electrophoresis and Western blotting.

This prestained protein MW marker is designed for monitoring the progress of SDS-polyacrylamide gel electrophoresis, for assessing transfer efficiency onto PVDF, nylon and nitrocellulose membranes, and for estimating the approximate size of separated proteins that have been made visible with gel stains or Western blot detection reagents. Three different chromophores (blue, orange, green) are bound to the different component proteins, producing a brightly colored ladder with an easy-to-remember pattern. The Spectra Multicolor High Range Protein Ladder is ready to use: no heating, further dilution or addition of a reducing agent is required.

Highlights:

• Size range – six proteins and peptides spanning 1.7 to 40kDa

• Multicolor – three different colors for unambiguous band-size assignment

• Ready-to-use – supplied in a loading buffer for direct loading on gels; no need to boil

• Sharp bands – color-coded bands of similar intensity for easy visualization

• Quality tested – each lot evaluated by SDS-PAGE and Western blotting

• Membrane-compatible – colored bands transfer to membranes for Western blotting

Includes:

Dye-stained proteins in 62.5mM Tris-H³PO⁴ (pH 7.5 at 25°C), 1mM EDTA, 2% SDS, 10mM DTT, 1mM NaN³ and 33% glycerol.

Recommended for:

Monitoring of protein migration during SDS-PAGE; Verifying Western transfer efficiency; Approximate sizing of proteins on SDS-polyacrylamide gels and Western blots; Locating a protein of interest for excision from an unstained preparative gel

Specifications

• Content And Storage: Contents: one vial of 250 μL
  
  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
  
  Storage: Upon receipt store at -20°C
  
  
• Detection Method: Colorimetric, NIR Fluorescence (700 nm), RGB Fluorescence (555 nm)
• Number of Markers: 6
• Ready to Load: Yes
• Recommended Applications: Tricine gels
• Size Range: 1.7 to 40 kDa
• For Use With (Application): Tricine gels
• Gel Compatibility: Bolt™ Bis-Tris Plus Gels, Novex™ Tricine Gels, Novex™ Tris-Glycine Gels, NuPAGE™ Bis-Tris Gels, NuPAGE™ Tris-Acetate Gels, SDS-PAGE Gels
• Molecular Weight (g/mol): 40, 25, 15, 10, 4.6, 1.7 kDa
• Quantity: 250 μL
• Shipping Condition: Approved for shipment on Wet or Dry Ice
• Product Line: Spectra
• Product Type: Protein Ladder
• Stain Type: Blue, Orange, Green
• System Type: Western Blotting, SDS-PAGE

Frequently Asked Questions (FAQs)

Why do Thermo Scientific prestained protein ladders not show the real protein sizes?

Coupling of chromophores to proteins affects the apparent molecular weight of proteins in SDS-PAGE relative to unstained standards. The apparent molecular weight of prestained protein standards is calibrated in the classical TRIS glycine-SDS Laemmli system, however prestained proteins may have different mobility in other electrophoresis buffer and gel systems. It should also be noted that the sizing of proteins by gel electrophoresis does not give an exact value and depends on the protein sequence and post-modification.

The upper bands of the ladder are missing. What could be the reason?

The upper bands of the ladder may be degraded by proteases. Ladder, gel, buffer, pipettes, pipette tips, or equipment can be contaminated by proteases during usage. A general recommendation would be to avoid working with proteases in the same room. We would recommend preparing fresh solutions, cleaning the equipment, and using clean pipettes and tips. If the ladder itself is contaminated, please use a new tube of the ladder.

Do the proteins in Thermo Scientific protein ladders have a His-Tag or would otherwise react with an anti-His-Tag antibody?

No, proteins in Thermo Scientific protein ladders are not His tagged. However, non-specific interaction between the ladder proteins and primary or secondary antibodies is possible and some His-Tag detection systems, such as Thermo Scientific 6xHis Protein Tag Stain Reagent Kit, show non-specific interaction. The protein ladder bands are more readily detected when using high antibody concentrations. The non-specific cross-reactivity is difficult to predict, it often has a different pattern dependent on the antibodies used in each individual experiment. The most general way to handle this problem would be to use lower concentrations of antibodies and to use lower amount of protein ladders. It may also be useful to leave one empty well between the ladder and the sample to overcome a possible leakage of the signal to the nearby sample lane.

Can Thermo Scientific protein ladders be detected by Strep-Tactin conjugates?

PageRuler Unstained protein ladders can be detected directly on Western blots by using Strep-Tactin conjugates or an antibody against the Strep-tag II sequence. All PageRuler and Spectra ladder proteins contain an integral Strep-tag II sequence, however the prestained ladders cannot be detected by Strep-Tactin conjugates.

Why are the PageRuler and Spectra ladder bands detected with antibodies against eukaryotic proteins?

All PageRuler and Spectra ladder bands are recombinant prokaryotic proteins purified from E. coli cells. None of them are related to eukaryotic proteins, however this cannot exclude the possibility that the ladder proteins may possess an epitope that is cross-reactive with the antibody used.

Why is non-specific binding detected after Western blot?

Protein ladder bands can sometimes be detected with chemiluminescent techniques due to non-specific interactions of ladder proteins with either primary or secondary antibodies (or with both). The ladder bands are only rarely detected by chromogenic substrates. The extremely high sensitivity of the chemiluminescent assays is needed to see the bands, so the actual degree of cross-reactivity is low. The non-specific cross-reactivity is difficult to predict, it often has a different pattern depending on the antibodies used. If antibodies recognize a linear epitope, the cross-reactivity may be due to sequence homology. If antibodies react with a denaturation-resistant conformational epitope it could be impossible to identify the exact reason for detected cross-reactivity. The most general way to handle this problem would be to use lower concentrations of antibodies.

Do Thermo Scientific protein ladders have glycosylated proteins?

No. Thermo Scientific protein ladders contain a mix of recombinant prokaryotic proteins purified from E. coli cells. E. coli does not have native glycosylation pathways, so none of the ladder proteins are glycosylated.

What are the concentrations of the individual proteins in the Thermo Scientific protein ladders? Is it possible to use the ladders as a standard for protein quantification?

Thermo Scientific ladders are not designed for protein quantification. For quantification, we would recommend to use a protein of known concentration as a reference.

Do you have protein ladders for fluorescence imaging?

Yes, PageRuler Prestained NIR Protein Ladder (Cat. No. 26635) contains proteins that are blue-stained and fluor-labeled for near-IR fluorescent visualization and protein sizing following electrophoresis.

Could a protein ladder be used on 6% Tris-glycine SDS-PAGE, although 4-20% gel is recommended? What effect would it have if the ladder is run on such a low concentration gel?

Protein ladders can be run on lower percentage gels than we recommend, however it should be expected that several bands of lower molecular weight proteins will run out of the gel if the gel is run until the dye front reaches the bottom of the gel. In case of shorter electrophoresis time it is also possible that some lower bands will not separate and will be covered by the dye front. In addition, lower molecular weight protein bands may look diffused. The lower percentage gels can be used in cases when the customer is interested in visualization of large proteins only.

What gel running buffer should I use: Tris-glycine, Tris-tricine, or Tris-acetate?

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).

When should unstained or prestained ladder/marker be used?

Prestained ladders/markers are recommended for approximate determination of molecular weight and for monitoring the progress of the electrophoresis run and the efficiency of protein transfer to membranes during Western blotting procedures. Unstained ladders/markers are used for precise determination of molecular weights in any denaturing buffer system.

Can you provide the precise concentration of the proteins in Thermo Scientific ladders?

We do not provide the exact or approximate concentration of proteins in Thermo Scientific protein ladders, and they are not meant to be used for quantifying the protein concentration of a band. For densitometry assessment, we recommend loading a known amount of a protein standard and determining the linear range according to the gel or membrane stain used.

I used one of your Thermo Scientific Spectra prestained protein ladders for a western transfer and got very poor transfer onto the membrane. What possibly went wrong?

Here are possible causes and solutions:

- Not enough volume of ladder loaded on the gel: Load an appropriate volume of the ladder onto the gel. Here are our recommendations:
--- Mini-gel: 5 µL per well (0.75-1.0 mm thick) or 10 µL per well (1.5 mm thick)
--- Large gel: 10 µL per well (0.75-1.0 mm thick) or 20 µL per well (1.5 mm thick)
- Incomplete or poor transfer: Optimize transfer conditions

I used one of your Thermo Scientific Spectra prestained protein ladders and did not get good separation of the bands. What could have happened?

Here are possible causes and solutions:

Here are possible causes and solutions:

- Ladder was boiled: Discard boiled aliquot.
- Too much volume of ladder used: Add less volume or dilute the ladder in protein loading buffer prior to use.

I used one of your pre-stained protein standards for a western transfer and I noticed that the intensity of the band faded from the membrane during the transfer process. Why is this?

The fading is most likely due to detergent in the western blocking/washing solutions that can remove some of the proteins from the membrane. The dye itself will not wash off of the proteins because it is covalently bound. We have found that smaller pore size membranes retain the proteins better during blocking and wash procedures, and hence recommend use of 0.2 µm instead of 0.45 µm membranes for best resolution and protein retention. 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.

I used one of your protein standards for a western transfer and noticed that some of the lower-molecular weight protein bands passed through the membrane. How can I resolve this issue?

- Decrease voltage, current or length of transfer time
- Make sure that the methanol concentration in the transfer buffer is proper; use a methanol concentration of 10-20% methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane.
- Make sure that the SDS concentration (if added) in the transfer buffer is proper, don't use more than 0.02-0.04% SDS. Using too much SDS can prevent binding of proteins to the membrane.
- Check the pore size of the membrane and the size of the target protein. Proteins smaller than 10 kDa will easily pass through a 0.45 µm pore size membrane. If proteins smaller than 10 kDa are of interest, it would be better to use a 0.2 µm pore size membrane.

I used one of your protein standards for a western transfer and noticed that some of the higher-molecular weight bands transferred very poorly to the membrane. Can you offer some tips?

- Increase voltage, current or length of time for transfer
- SDS in the gel and in the SDS-protein complexes promotes elution of the protein from the gels but inhibits binding of the protein to membranes. This inhibition is higher for nitrocellulose than for PVDF. For proteins that are difficult to elute from the gel such as large molecular weight proteins, a small amount of SDS may be added to the transfer buffer to improve transfer. We recommend pre-equilibrating the gel in 2X Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X transfer buffer containing 10% methanol and 0.01%SDS.
- Methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane, but has some negative effects on the gel itself, leading to a decrease in transfer efficiency. It may cause a reduction in pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral. Make sure that the methanol concentration in the transfer buffer is not more than 10-20% and that high-quality, analytical grade methanol is used.

I used one of your pre-stained standards on a Tris-Glycine gel and noticed that the molecular weights of the proteins were different than on a NuPAGE Bis-Tris gel. What is the reason for this?

Pre-stained standards have a dye that is covalently bound to each protein that will result in the standard migrating differently in different buffer systems (i.e., different gels). As a result, using a pre-stained standard for molecular weight estimation will only give the apparent molecular weight of the protein. Pre-stained standards may be used for molecular weight approximation, confirming gel migration and estimating blotting efficiency but for accurate molecular weight estimation, an unstained standard should be used.

I used one of your protein standards and am seeing some extra bands in the lane. Can you offer some suggestions?

- While loading, take care to make sure that there is no cross-contamination from adjacent sample lanes.
- Make sure that the correct amount of standard is loaded per lane. Loading too much protein can result in extra bands and this is a problem especially with silver-stained gels.
- Improper storage of the standard or repeated freeze/thawing can result in protein degradation.

I used one of your protein standards and the bands look non-distinct and smeary. What should I do?

Here are some suggestions:

- Make sure that the correct amount of standard is loaded per lane. Loading too much protein can cause smearing and this is a problem especially with silver stained gels.
- Bands will not be as well resolved in low percentage gels. Try using a higher percentage gel.
- If the bands look smeary and non-distinct after a western transfer/detection, this may be due to the antibody being too concentrated. Follow the manufacturer's recommended dilution or determine the optimal antibody concentration by dot-blotting.

A couple of bands in my protein standard are missing on the gel. Can you help me troubleshoot?

Here are some suggestions:

- Check the gel type/percentage of the gel that was used. Depending on the gel type and/or percentage, all the bands may not be seen. For example, the smallest bands of the protein standard may not resolve on a very low percentage gel whereas the higher molecular weight bands may not resolve on a high percentage gel.
- Check the expiration date on the protein standard. Expired lots may result in faded or missing bands due to protein degradation.
- Check the storage conditions for the protein standard. Improper storage conditions will compromise the stability of the proteins in the standard.
- Make sure that the protein standard was not heated/boiled prior to loading on the gel. Our protein standards are ready to load and we do not recommend heating/boiling them as this may cause degradation of proteins in the standard.

Are any animal-derived proteins present in the Spectra Protein Ladders?

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

What is the composition of the Spectra Multicolor Low Range Protein 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.

What are the storage conditions and shelf life for the Spectra Protein Ladders?

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.

Do protein standards run differently on a Zymogram gel compared to a regular Tris-Glycine gel?

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.

Can I use any of your protein standards for estimation of protein quantity (protein quantitation)?

Our protein standards are not designed for protein quantitation.

Can I use your prestained standards for native gel electrophoresis?

We do not recommend using our prestained standards for native gel electrophoresis since they are already denatured (in SDS sample buffer) and pre-reduced (by a proprietary method), and will not resolve well in under native conditions.

Can I use a prestained protein ladder to estimate the molecular weight of my protein?

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.

What is the recommended gel loading volume for your protein standards?

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

Do I need to boil your protein standards before loading on the gel?

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

Do I have to add reducing agent to your protein standards?

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

Safety and Handling

WARNING: Cancer - www.P65Warnings.ca.gov

For Research Use Only. Not for use in diagnostic procedures.