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Introduction
The MagicMark™ XP Western Protein Standard allows direct visualization of protein standard bands on a blot without the need for protein modification or special detection reagents. MagicMark™ XP Western Standard proteins are expressed in E. coli from a construct containing repetitive units of a fusion protein forming the size variation and an IgG binding site.
The important features of standard are listed below:
Specifications
The important features of standard are listed below:
- MagicMark™ XP consists of nine recombinant proteins in the range of 20–200 kDa
- Suitable for Western blotting and molecular weight estimation
- Provided in a ready-to-use format
- Visualized with alkaline phosphatase or peroxidase conjugated antibody using chromogenic, chemiluminescent, or fluorescent substrates
- Visualized also with SimplyBlue™ SafeStain or other Coomassie® stains on SDS-PAGE gels
Specifications
- Contents: 250 µl MagicMark™ XP Western Protein Standard
- Storage Buffer: 125 mM Tris-HCl, pH 6.8; 10 mM DTT; 17.4% glycerol; 3% SDS; and 0.025% bromophenol blue
- Storage: Store at -20°C. To avoid repeated freezing and thawing, aliquot in small volumes and store.
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Directions for Use
The MagicMark™ XP Western Protein Standard is supplied in a ready-to-use format. There is no need to heat or reduce the standard prior to loading.
- Load 2-10 µl of the standard on an appropriate SDS-PAGE mini-gel. We recommend testing different amounts of the standard to determine the optimal amount of standard to use under your experimental conditions.
- Load your samples and perform electrophoresis.
The amount of standard will depend on the binding affinity of MagicMark™ for your antibody species and sensitivity of your detection system.
Protocol - Blotting and Staining
Blotting
The MagicMark™ XP Western Standard proteins may not align with pre-stained or unstained markers.
Staining
Stain the gel with SimplyBlue™ SafeStain or other Coomassie® stains and then destain the gel.
Pre-mixing with SeeBlue® Pre-stained Standard
To monitor the electrophoresis run, pre-mix 10 µl MagicMark™XP Western Standard with 5 µl SeeBlue® Pre-stained Standard (catalog no. LC5625).
- Transfer proteins to a suitable membrane.
- Perform the blocking step, primary antibody incubation step, and (if necessary) secondary antibody incubation step with the blot using a method of choice.
- Visualize proteins using a colorimetric, chemiluminescent, or fluorescent detection system using the manufacturer’s recommendations. After detection, you should observe the protein standard bands.
The MagicMark™ XP Western Standard proteins may not align with pre-stained or unstained markers.
Staining
Stain the gel with SimplyBlue™ SafeStain or other Coomassie® stains and then destain the gel.
Pre-mixing with SeeBlue® Pre-stained Standard
To monitor the electrophoresis run, pre-mix 10 µl MagicMark™XP Western Standard with 5 µl SeeBlue® Pre-stained Standard (catalog no. LC5625).
Pre-mixing with SeeBlue® Pre-stained Standard
To monitor the electrophoresis run, pre-mix 10 µl MagicMark™XP Western Standard with 5 µl SeeBlue® Pre-stained Standard (catalog no. LC5625).
Affinity of MagicMark™XP Standard to Antibodies
| Species | Affinity of MagicMark™ |
| Human, Horse, Cow | ++++ |
| Pig, Rabbit | +++ |
| Goat, Sheep, Hamster, Guinea Pig, Rat, Mouse | ++ |
| Chicken | + |
Example
5 µl of MagicMark™ XP Western Protein Standard was loaded on a NuPAGE® Novex 4-12% Bis-Tris Gel (A) or a Novex® 4-20% Tris-Glycine gel (B), blotted onto a nitrocellulose membrane, and detected using the WesternBreeze® Anti-Rabbit Chemiluminescent Kit.
Troubleshooting
Review the information below to troubleshoot your experiments with MagicMark™ Western Standard. For troubleshooting Western blotting and detection, refer to the manufacturer’s recommendations.
| Problem | Cause | Remedy |
|---|---|---|
| Weak or no signal | Detection reagents not functional | Verify that the detection reagents are working well. Optimize the antibody concentration to obtain best results. |
| Low amount of standards loaded | Load higher amount of the standard on the gel. | |
| Poor or incomplete transfer | Optimize Western transfer. | |
| Enzyme-conjugated antibodies may not bind efficiently with MagicMark™ proteins | Use unconjugated primary antibody, followed by the addition of enzyme-conjugated secondary antibody. Note: The Anti-myc-AP/HRP and Anti-V5-AP/HRP Antibodies from Invitrogen do not bind to MagicMark™ proteins. | |
| Smeary, non-distinct bands | Overloading | Decrease amount of standard loaded. |
| Antibody is too concentrated | Follow the manufacturer’s recommended dilution or determine the optimal antibody concentration by dot-blotting. |
LC5600.pps MAN0001518 12-May-2010
