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Invitrogen™ HiMark™ Pre-stained Protein Standard

Catalog No. LC5699
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LC5699 250 μL
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Catalog No. LC5699 Supplier Invitrogen™ Supplier No. LC5699
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Description

Includes

250μL of HiMark™ Pre-Stained HMW Protein Standard; Storage Buffer: Tris-HCl, Formamide, SDS, Phenol Red

Designed for analysis of high molecular weight proteins on NuPAGE™ Novex™ Tris-Acetate gels

  • Comprehensive - contains markers with a wide range of high molecular weights, providing nine protein bands in the range of 30 to 460kDa
  • Convenient - a ready-to-use formulation eliminates the need to heat, reduce, or add sample buffer prior to use
  • Efficient - allows easy visualization of electrophoresis and rapid evaluation of Western transfer efficiency
  • Compatible - intended for use with NuPAGE Novex 3 to 8% and 7% Tris-Acetate gels under denaturing conditions
  • Can also be used with NuPAGE Novex 4-12% Bis-Tris Gels with NuPAGE MOPS Running Buffer and Novex 4% Tris-Glycine Gel, however to obtain best results with HMW proteins, always use NuPAGE Novex Tris-Acetate Gels
  • Gel Compatibility: NovexTris-Glycine Gels, NuPAGE Tris-Acetate Gels

HiMark™ High Molecular Weight Protein Standard 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: Dry Ice

Specifications

Content And Storage Contents: 250 μL of HiMark™ Pre-Stained HMW Protein Standard
Storage Buffer: Tris-HCl, Formamide, SDS, Phenol Red
Storage:Store at -20°C
Detection Method Colorimetric
Number of Markers 9
Ready to Load Yes
Recommended Applications Tris-acetate gels
Size Range 30 to 460 kDa
For Use With (Application) Tris-acetate gels
Gel Compatibility Novex™ Tris-Glycine Gels, NuPAGE™ Tris-Acetate Gels
Molecular Weight (g/mol) 460, 268, 238, 171, 117, 71, 55, 41, 31 kDa
Quantity 250 μL
Shipping Condition Dry Ice
Product Line HiMark
Product Type Protein Ladder
Stain Type Blue, Pink
System Type Western Blotting, SDS-PAGE
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Frequently Asked Questions (FAQs)

Can I use the HiMark High Molecular Weight Protein Standard with your NuPAGE Bis-Tris or NuPAGE Tris-Glycine gels?

Yes, the HiMark High Molecular Weight Protein Standard can be used with our NuPAGE gels, though we would recommend using our NuPAGE Tris-Acetate gels for best results when separating high moleuclar weight proteins.

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 use the HiMark Prestained Protein Standard with NuPAGE Bis-Tris gels and Tris-Glycine gels?

The HiMark Prestained Protein Standard can be used with NuPAGE Invitrogen 4-12% Bis-Tris Gels (with NuPAGE MOPS SDS Running Buffer) and Invitrogen 4% Tris-Glycine Gel (with Tris-Glycine SDS Running buffer). However, to obtain the best results with high molecular weight proteins, we recommend using the HiMark Prestained Protein Standard with NuPAGE Invitrogen Tris-Acetate Gels (with Tris-Acetate SDS buffer system).
I am planning to run a NuPAGE Tris-Acetate gel to resolve a 150 kDa protein. Which of your prestained protein standards should I use?

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.
What are the storage conditions and shelf life for the HiMark Prestained Protein Standard?

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

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