Invitrogen™ Mark12™ Unstained Standard

Product Code.	Quantity	unitSize
10154962	1 mL	1mL

Product Code. 10154962 Supplier Invitrogen™ Supplier No. LC5677

Description

Includes

1.0 mL (200 applications of 5μL each) provided in a plastic vial Loading Buffer consists of Tris-HCl, Glycerol, SDS, Phenol Red, and Coomassie Blue G-250

Allows the closest estimation of molecular weight of your sample protein over a wide molecular weight range.

Mark12 Unstained Standard is supplied ready-to-use with either NuPAGE™, tris-glycine, or tricine gels. Coomassie™, silver, or other protein staining methods are required to visualize this standard.

• 12 polypeptides resolved into sharp, tight bands in the range of 2.5 to 200kDa
• Ideal molecular weight estimation
• A ready-to-use format no mixing or reducing required
• Gel Compatibility: Bolt Bis-Tris Plus Gels, NovexTricine Gels, NovexTris-Glycine Gels, NuPAGE Bis-Tris Gels, NuPAGE Tris-Acetate Gels, SDS-PAGE Gels

This standard is pre-reduced and does not contain reducing reagents in the loading buffer.

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

Order Info

Shipping Condition: Wet Ice

Specifications

• Content And Storage: 1.0 mL (200 applications of 5 μL each) provided in a plastic vial. Loading Buffer consists of Tris-HCl, Glycerol, SDS, Phenol Red, and Coomassie™ Blue G-250.
  
  Store at 4°C.
• Number of Markers: 12
• Ready to Load: Yes
• Size Range: 2.5 to 200 kDa
• Gel Compatibility: Bolt™ Bis-Tris Plus Gels, Novex™ Tricine Gels, Novex™ Tris-Glycine Gels, NuPAGE™ Bis-Tris Gels, NuPAGE™ Tris-Acetate Gels, Novex™Tricine, SDS-PAGE Gels
• Molecular Weight (g/mol): 200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, 14.4, 6, 3.5, 2.5 kDa
• Quantity: 1 mL
• Shipping Condition: Wet Ice
• Product Line: Mark12
• Product Type: Protein Ladder
• Stain Type: Unstained
• System Type: Western Blotting, SDS-PAGE

Frequently Asked Questions (FAQs)

Can I use the Mark12 Unstained Protein marker to determine molecular weights in a gel filtration column?

This is not recommended. The buffer conditions in gel filtration are not compatible with this standard.

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.

What are the molecular weights and origins of the proteins in the Mark12 Unstained Standard?

The Mark12 Unstained Standard is suitable for NuPAGE, Tris-Glycine and Tricine gels. Here are the molecular weights and origins of the proteins in the Mark12 Unstained Standard:

- Myosin: 200 kDa; Rabbit muscle
- b-galactosidase: 116.3kDa; E. coli
- Phosphorylase B: 97.4 kDa; Rabbit muscle
- Bovine serum albumin: 66.3 kDa; Bovine serum
- Glutamic dehydrogenase: 55.4 kDa; Bovine liver
- Lactate dehydrogenase: 36.5 kDa; Porcine
- Carbonic anhydrase: 31.0 kDa; Bovine
- Trypsin inhibitor: 21.5 kDa; Soybean
- Lysozyme: 14.4 kDa; Egg white
- Aprotinin: 6.0 kDa; Bovine lung
- Insulin B chain: 3.5* kDa; Bovine pancreas
- Insulin A chain: 2.5*kDa; Bovine pancreas

Note: The 3.5 kDa and 2.5 kDa bands are visible only on NuPAGE Bis-Tris gels run using 1X MES SDS Running buffer.

What are the storage conditions and shelf life for the Mark12 Unstained Standard?

We recommend storing the Mark12 Unstained Standard at 4 degrees C and it is stable until the expiration date 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.