Thermo Scientific™ Spectra™ Multicolor High Range Protein Ladder

Catalog No.	Quantity
26-625-X4	8 x 250 μL
PI26625	2 x 250 μL

Catalog No. 26-625-X4 Supplier Thermo Scientific™ Supplier No. 26625X4

Description

A mixture of eight blue-, green- and orange-stained proteins (40 to 300kDa) for use as size standards for high MW proteins in gel electrophoresis and Western blotting.

Thermo Scientific™ Spectra Multicolor High Range Protein Ladder is a mixture of eight (8) blue-, green and orange-stained proteins (40 to 300kDa) for use as size standards for high MW 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 – eight proteins spanning 40 to 300kDa

• 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: eight vials of 250 μL each
  
  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: 8
• Ready to Load: Yes
• Size Range: 40 to 300 kDa
• Molecular Weight (g/mol): 300, 250, 180, 130, 100, 70, 50, 40 kDa
• Quantity: 8 x 250 μL
• Product Line: Spectra
• Product Type: Protein Ladder
• Stain Type: Blue, Orange, Green
• System Type: SDS-PAGE

Frequently Asked Questions (FAQs)

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 High Range Protein 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.

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