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:

  • 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.
 
  1. 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.
  2.  
    The amount of standard will depend on the binding affinity of MagicMark™ for your antibody species and sensitivity of your detection system.
     


  3. Load your samples and perform electrophoresis.
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Protocol - Blotting and Staining

Blotting
 
  1. Transfer proteins to a suitable membrane.

  2. Perform the blocking step, primary antibody incubation step, and (if necessary) secondary antibody incubation step with the blot using a method of choice.

  3. 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).

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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).

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Affinity of MagicMark™XP Standard to Antibodies

Species
Affinity of MagicMark™
Human, Horse, Cow
++++
Pig, Rabbit
+++
Goat, Sheep, Hamster, Guinea Pig, Rat, Mouse
++
Chicken
+
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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.

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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.

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LC5600.pps  MAN0001518   12-May-2010