MagicMark™ XP Western Protein Standard, 250 μL - 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.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

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.

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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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

- 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

- 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.

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- 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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The exact protein amount in each band of the standard is proprietary. We do not recommend using this standard for quantitation purpose.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Here are the relative affinities of the MagicMark XP Western Protein Standard to IgG antibodies from different species:

Human, Horse, Cow: ++++
Pig, Rabbit: +++
Goat, Sheep, Hamster, Guinea Pig, Rat, Mouse: ++ Chicken: +

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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.

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Each protein in the MagicMark XP Western Protein Standard contains an IgG binding site, allowing direct visualization with the same antibody-conjugate reagents used to detect the target protein. The proteins in the standard will not bind to IgM antibodies.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The MagicMark XP protein bands can be visualized with the same antibody-conjugate reagents used to detect the target protein, using either colorimetric or chemiluminescent methods. MagicMark XP standard can be used for molecular weight estimation of proteins. Each protein in the MagicMark XP Western Protein Standard contains an IgG binding site. The proteins in the standard will not bind to IgM antibodies.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The MagicMark XP Western Protein Standard is designed for molecular weight estimation of proteins directly on western blots. Each protein in the standard contains an IgG binding site, allowing direct visualization with the same antibody-conjugate reagents used to detect the target protein on blots. Colorimetric, chemiluminescent, or fluorescent detection methods can be used for analysis. MagicMark XP Western Protein Standard is compatible with NuPAGE and Tris-Glycine gels. Here are the molecular weights of the proteins in the MagicMark XP Western Protein Standard:

- Band 1: 220 kDa
- Band 2: 120 kDa
- Band 3: 100 kDa
- Band 4: 80 kDa
- Band 5: 60 kDa
- Band 6: 50 kDa
- Band 7: 40 kDa
- Band 8: 30 kDa
- Band 9: 20 kDa


Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

We recommend storing the MagicMark XP Western Protein Standard -20 degrees C in aliquots to avoid repeated freezing and thawing. It is stable for 4 months when properly stored.

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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.

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Our protein standards are not designed for protein quantitation.

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Please find this information in the respective manuals for the individual protein standards.

Find additional tips, troubleshooting help, and resources within our Protein Standards and Ladders Support Center.

Our protein standards are ready to load. We do not recommend heating them as this may cause protein degradation.

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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.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.