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Invitrogen™ Novex™ Sharp Pre-stained Protein Standard

Catalog No. LC5800
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LC5800 2 x 250 μL
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Catalog No. LC5800 Supplier Invitrogen™ Supplier No. LC5800
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Description

Includes

Novex Sharp Pre-stained Protein Standards provided as 2 x 250μL (total of 50 applications of 10μL each) of ready-to-use standardmixture Novex Sharp Pre-Stained standard loading buffer consists of 65 mM Tris pH 6.5, 30% glycerol, 2% SDS, and 2.5 mM ETDA

Designed for accurate, easy, and convenient molecular weight estimation of a wide range of molecular weight proteins during SDS-PAGE and Western blotting

  • Accurate - sharp bands for the accurate estimation of molecular weight
  • Unambiguous - each band in the standard is pre-stained with a unique color for easy interpretation of results
  • Comprehensive - a wide range of molecular weight bands consisting of 12 proteins in the range of 3.5 to 260kDa
  • Convenient - a ready-to-use formulation eliminates the need to heat, reduce, or add sample buffer prior to use
  • Adaptable - suitable for most gel types, recommended for use with Novex™ NuPAGE™, Tris-Glycine, and Tricine gels
  • Gel Compatibility: Bolt Bis-Tris Plus Gels, NovexTricine Gels, NovexTris-Glycine Gels, NuPAGE Bis-Tris Gels

The Novex Sharp Protein Standard is also available in an unstained format.

1D Gel Electrophoresis, Protein Gel Electrophoresis, Protein Gel Staining and Imaging, Proteins, Expression, Isolation and Analysis, Western Blotting

Order Info

Shipping Condition: Approved for shipment on Wet or Dry Ice

Specifications

Content And Storage Novex™ Sharp Pre-stained Protein Standards are provided as 2 x 250 μL (total of 50 applications of 10 μL each) of ready-to-use standard mixture. The Novex™ Sharp Pre-Stained standard loading buffer consists of 65 mM Tris pH 6.5, 30% glycerol, 2% SDS, and 2.5 mM ETDA.

Store at -20°C.
Detection Method Colorimetric
Number of Markers 12
Ready to Load Yes
Size Range 3.5 to 260 kDa
Gel Compatibility Bolt™ Bis-Tris Plus Gels, Novex™ Tricine Gels, Novex™ Tris-Glycine Gels, NuPAGE™ Bis-Tris Gels
Molecular Weight (g/mol) 260, 160, 110, 80, 60, 50, 40, 30, 20, 15, 10, 3.5 kDa
Quantity 2 x 250 μL
Shipping Condition Approved for shipment on Wet or Dry Ice
Product Line Novex
Product Type Protein Ladder
Stain Type Blue, Yellow, Pink
System Type Western Blotting, SDS-PAGE
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Frequently Asked Questions (FAQs)

Why can I not see the 3.5 kDa band of the Invitrogen Sharp Pre-Stained Standard on my NuPAGE gels?

The 3.5 kDa band is visible only on NuPAGE gels with 1X MES SDS running buffer.
What are the different molecular weight estimations for the Invitrogen Sharp Pre-Stained standard on the different Invitrogen gel types?

Invitrogen Sharp Pre-Stained Standard molecular weight estimations are the same in NuPAGE Bis-Tris, NuPAGE Tris-Acetate, Invitrogen Tris-Glycine, and Tricine Gels. The appearance/migration of the protein bands may differ depending upon the gel type and running conditions
What are the recommended protocols for preparing Invitrogen protein standards to load on a gel?

Invitrogen protein standards do not require any additional preparation. Thaw protein standards at room temperature, vortex to mix well, and load.
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.

Can I combine MagicMark XP Western Protein standard with one of your prestained standards?

You can combine 5 µL MagicMark XP Standard with 5 µL Invitrogen 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.

What are the apparent molecular weights of the proteins in the Invitrogen Sharp Prestained Protein Standard in the different gel types?

The Invitrogen 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 Invitrogen 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. Please see figure on this webpage (http://www.thermofisher.com/order/catalog/product/LC5800?ICID=search-product) 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.
Can I use the Invitrogen Sharp Prestained Standard on zymogram gels?

The Invitrogen 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.
What are the origins of the proteins in the Invitrogen Sharp Prestained Protein Standard?

The proteins in the Invitrogen Sharp Prestained Protein Standard are synthetic fusion proteins and their origins are proprietary. The 10, 20, 60 and 80 kDa proteins in the Invitrogen Sharp Prestained Standard have C-terminal 6xHis tags and hence are shown to cross react with the anti-His C-term antibody.
What are the storage conditions and shelf life for the Invitrogen Sharp Prestained Protein Standard?

We recommend storing the Invitrogen Sharp Prestained Protein Standard at -20 degrees C. It is stable for 12 months when properly stored.
I have been using the MultiMark Multi-Colored Standard and it has been discontinued. Is there an alternative product?

Yes, we recommend the Invitrogen 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.
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.
Can I use the Invitrogen Sharp Pre-Stained Standard with the NativePAGE system?

No, most of our protein standards (including the Invitrogen Sharp prestained and unstained standards) are already denatured and reduced, and will not resolve well in a native gel. For NativePAGE electrophoresis, we offer the NativeMARK Unstained Protein Standard (Cat. No. LC0725). It is a ready-to-use protein marker that allows for molecular weight estimation of proteins using native gel electrophoresis.

Please note that even with a native standard, molecular weight estimation in native electrophoresis is very inaccurate, as gel migration can be significantly affected by differing charge and conformation of individual proteins. For more accurate estimations, use SDS-PAGE separation or mass spectrometry analysis.

Which pre-stained protein standards are compatible with Bolt Bis-Tris Plus gels?

SeeBlue prestained standard, SeeBlue Plus 2 prestained standard and Invitrogen Sharp prestained standard are compatible with Bolt Bis-Tris Plus gels. Their migration is the same as that in NuPAGE gels with the corresponding buffer.

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