Invitrogen™ NativeMark™ Unstained Protein Standard

Catalog No.	Quantity
LC0725	5 x 50 μL

Catalog No. LC0725 Supplier Invitrogen™ Supplier No. LC0725

Description

Includes

Supplied in storage buffer (Bis/Tris-HCl, pH 7.0; NaCl; glycerol; Ponceau S)

Ready-to-use protein standard designed for molecular weight estimation of proteins using native gel electrophoresis

Electrophoresis under native (nondenaturing) conditions maintains the native protein conformation, cohesion of subunits, and biological activity. During native electrophoresis, proteins are separated based on their charge to mass ratios.

• Versatile: recommended for use with Novex NativePAGE™ Bis-Tris, gradient Tris-Glycine, or NuPAGE Novex Tris-Acetate gels
• Comprehensive: contains markers with a wide range of high molecular weights, providing 8 protein bands in the range of 20–1,200 kDa
• Convenient: no need to add sample buffer, just load and run
• Compatible: can be visualized using Coomassie™, silver, or fluorescent stains after electrophoresis, or with Ponceau S, Coomassie, or other membrane stains after Western transfer
• Gel Compatibility: NativePAGE Gels, NovexTris-Glycine Gels, NuPAGE Bis-Tris Gels, NuPAGE Tris-Acetate Gels

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

Order Info

Shipping Condition: Dry Ice

Specifications

• Content And Storage: The NativeMark™ Unstained Protein Standard is supplied in storage buffer (Bis/Tris-HCl, pH 7.0; NaCl; glycerol; Ponceau S).
  
  Store at -20°C.
• Number of Markers: 8
• Ready to Load: Yes
• Recommended Applications: Native gels
• Size Range: 20 to 1,200 kDa
• For Use With (Application): Native gels
• Gel Compatibility: Novex™ Tris-Glycine Gels, NuPAGE™ Bis-Tris Gels, NuPAGE™ Tris-Acetate Gels, Novex™Tris-Glycine Gels, NativePAGE™ Gels, NuPAGE™ Tris-Acetate Gels
• Molecular Weight (g/mol): 1236, 1048, 720, 480, 242, 146, 66, 20 kDa
• Quantity: 5 x 50 μL
• Shipping Condition: Dry Ice
• Product Line: NativeMARK
• Product Type: Protein Ladder
• Stain Type: Unstained
• System Type: Native-PAGE

Frequently Asked Questions (FAQs)

After staining an IEF gel, what are reasons why the protein samples do not appear but the IEF protein standard (marker) does?

1) If the protein has a pI greater that 8.5, it may be able to be resolved on a pH 3-10 gel.
2) The protein may be insoluble.
3) There may be protein loss if the fixative is too weak in the fixation step prior to staining.
4) Your sample load may be too low.

Can I use your Protein standards for native gel applications?

We only offer one protein standard for native gel electrophoresis - NativeMark Unstained Protein Standard (Cat. No. LC0725) which is a ready-to-use protein marker for the estimation of the molecular weight of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE Invitrogen Tris-Acetate Gels. It contains 8 proteins in the range of ~20-1200 kDa that can be visualized by Coomassie or silver staining.

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 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 NativeMark Unstained Protein Standard for denaturing electrophoresis applications?

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.

What are the origins of the IgM Hexamer and IgM Pentamer protein bands in the NativeMark protein standard?

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

What are the molecular weights of the proteins in the NativeMark Unstained Protein Standard?

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 are the molecular weights of the proteins in the NativeMark Unstained Protein Standard:

Band 1: IgM hexamer; 1236 kDa in NativePAGE 3-12% Bis-Tris, NuPAGE 3-8% Tris-Acetate, and 4-12% Tris-Glycine gels
Band 2: IgM pentamer; 1048 kDa in NativePAGE 3-12% Bis-Tris, NativePAGE 4-16% Bis-Tris, NuPAGE 3-8% Tris-Acetate, and 4-12% Tris-Glycine gels
Band 3: Apoferritin band 1; 720 kDa in NativePAGE 3-12% Bis-Tris, NativePAGE 4-16% Bis-Tris, NuPAGE 3-8% Tris-Acetate, and 4-12% Tris-Glycine gels
Band 4: Apoferritin band 2; 480 kDa in NativePAGE 3-12% Bis-Tris, NativePAGE 4-16% Bis-Tris, NuPAGE 3-8% Tris-Acetate, and 4-12% Tris-Glycine gels
Band 5: β-phycoerythrin*; 242 kDa in NativePAGE 3-12% Bis-Tris, NativePAGE 4-16% Bis-Tris, NuPAGE 3-8% Tris-Acetate, and 4-12% Tris-Glycine gels
Band 6: Lactate dehydrogenase; 146 kDa in NativePAGE 3-12% Bis-Tris, NativePAGE 4-16% Bis-Tris, NuPAGE 3-8% Tris-Acetate, and 4-12% Tris-Glycine gels
Band 7: Bovine serum albumin; 66 kDa in NativePAGE 3-12% Bis-Tris, NativePAGE 4-16% Bis-Tris, and 4-12% Tris-Glycine gels
Band 8: Soybean trypsin inhibitor; 20 kDa in NativePAGE 3-12% Bis-Tris, NativePAGE 4-16% Bis-Tris, and 4-12% Tris-Glycine gels

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

What are the storage conditions and shelf life for the NativeMark Unstained Protein Standard?

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.

I would like to run a NativePAGE gel. Which of your protein standards should I use?

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

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.

Which protein standard do you recommend using with gels run under native conditions?

We recommend using the NativeMark Unstained Protein Standard, Cat. No. LC0725.

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