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Invitrogen™ MagicMark™ XP Western Protein Standard
Description
Includes
250 μL of ready-to-use standard mixture is provided in a plastic vial; Loading buffer consists of 125 mM Tris-HCl (pH 6.8), 10 mM DTT, 17.4% Glycerol, 3% SDS, and 0.025% Bromophenol Blue
- MagicMark XP Western Protein Standard consists of nine recombinant proteins (20–220 kDa), each of which contains an IgG binding site
- The IgG binding site binds the primary or secondary antibody used for detection of the target protein, allowing direct visualization of the standard on the western blot
- The MagicMark XP Standard is compatible with most western kits and substrates (chemiluminescent, chromogenic, and fluorescent)
- The protein standard is supplied in a ready-to-use format for direct loading onto gels; no need to heat, reduce, or add sample buffer prior to use
- Store at -20°C
Applications
- Western blotting: detection of the nine unstained bands via the detection method used for the target protein. Compatible with chemiluminescent substrates and fluorescent secondary antibodies (not recommended for antibodies labeled with fluors in the 500–550 nm channel)
- Protein size estimation: protein sizing on SDS-polyacrylamide gels and on western blot using western blot detection method
Order Info
Shipping Condition: Dry Ice
Specifications
Specifications
| Content And Storage | 250 μL of ready-to-use standard mixture is provided in a plastic vial. Loading buffer consists of 125 mM Tris-HCl (pH 6.8), 10 mM DTT, 17.4% Glycerol, 3% SDS, and 0.025% Bromophenol Blue. Store at -20°C. |
| Detection Method | User defined detection system |
| Number of Markers | 9 |
| Ready to Load | Yes |
| Size Range | 20 to 220 kDa |
| Gel Compatibility | Bolt™ Bis-Tris Plus Gels, Novex™ Tricine Gels, Novex™ Tris-Glycine Gels, NuPAGE™ Bis-Tris Gels, NuPAGE™ Tris-Acetate Gels, SDS-PAGE Gels |
| Molecular Weight (g/mol) | 220, 120, 100, 80, 60, 50, 40, 30, 20 kDa |
| Quantity | 250 μL |
| Shipping Condition | Dry Ice |
| Product Line | MagicMark |
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Frequently Asked Questions (FAQs)
The Positope control protein (Cat. No. R90050) is designed to be used as a positive control in western blotting for a variety of antibodies. The control protein is a 53 kDa highly purified recombinant protein that contains seven epitope tags including the Xpress, c-myc, V5, His(C-term), HisG(N-term), Thioredoxin, and GFP.
The MagicMark XP Western Protein Standard (Cat. No. LC5602) consists of peptides that contain an IgG binding domain. These peptides can be detected with your regular antibodies and provide a way for you to get good molecular weight estimations without having to rely on transferred pre-stained standards.
MagicMark XP standards consist of a mixture of eight E. coli-expressed recombinant proteins ranging in size from 20 to 120 kDa. Each protein contains an IgG binding sequence from Protein G. This IgG binding site binds the primary or secondary antibody used to detect your target protein, allowing you to detect the MagicMark standard directly.
Here are some suggestions:
- Verify that the detection reagents are working well. Optimize the antibody concentration to obtain best results.
- Make sure that the amount of standard loaded on the gel is correct.
- Optimize the transfer conditions (current, voltage, transfer time).
- Enzyme-conjugated primary antibodies may not bind efficiently with the proteins in the MagicMark XP Western Protein Standard. We recommend using unconjugated primary antibody, followed by the addition of enzyme-conjugated secondary antibody.
Note: The Anti-myc-AP/HRP and Anti-V5-AP/HRP antibodies do not bind to MagicMark XP proteins.
- 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.
- 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.
For Research Use Only. Not for use in diagnostic procedures.
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