HiMark™ Unstained High Molecular Weight Protein Standard Cat. no. LC5688 Size 250 µL Store at −30°C to −10°C Pub. Part no. LC5688.pps MAN0001520 Rev. Date: 5 December 2011

Description The HiMark™ Unstained High Molecular Weight (HMW) Protein Standard allows you to accurately estimate the molecular weight of high molecular weight proteins on NuPAGE® Novex Tris-Acetate Gels with Tris-Acetate SDS buffer system.

The important features of the standard are:

• Consists of 9 protein bands in the range of 40–500 kDa

• Designed for use with NuPAGE® Novex 3-8% and 7% Tris-Acetate Gels under denaturing conditions

• Supplied in a ready-to-use format

• Visualized with Coomassie or silver staining

• Visualized also with Ponceau S, Coomassie, or other membrane stains after western transfer

Specifications Contents: 250 μL of HiMark™ Unstained HMW Protein Standard

Storage 250 mM Tris-HCl, pH 8.5; 0.5 mM EDTA; 50 mM DTT; Buffer: 10% glycerol; 2% LDS; 0.2 mM Coomassie G-250; 0.175 mM Phenol red

Storage: Store at −30°C to −10°C. Avoid repeated freezing and thawing

Stability: 6 months at −20°C

Product Use For research use only. Not for human or animal therapeutic or diagnostic use.

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Directions The HiMark™ Unstained HMW Protein Standard is supplied in a ready-to-use format. There is no need to heat or add reducing agent.

1. Thaw the standard at room temperature. Vortex gently to ensure the solution is homogenous. Note: If there is a precipitate in the standard, thaw at room temperature for 10–15 minutes and vortex to solubilize or warm the solution at 30°C (Do not heat at >37°C).

2. Load the ladder using the loading volumes listed below to obtain the best results. Use an appropriate percentage of NuPAGE® Novex Tris-Acetate Gel to resolve the proteins.

Gel Electrophoresis/Blotting

Mini-Gel (1.0-mm thick) 5 μL Mini-Gel (1.5-mm thick) 7 μL

3. After electrophoresis, we recommend staining the standard proteins with SilverQuest® Silver Staining Kit, SimplyBlue™ SafeStain, or Coomassie stain. Avoid using the SilverXpress® Silver Staining Kit.

Blotting: To obtain higher transfer efficiency during the transfer of high molecular weight proteins, avoid using methanol in the transfer buffer. After transfer, you may stain the standard proteins on the membrane with Ponceau S, Coomassie, or any membrane stain of choice.

Note: The HiMark™ Unstained HMW Protein Standard is designed for use with NuPAGE® Novex Tris-Acetate Gels. Using the standards with NuPAGE® Novex Bis-Tris Gels or Tris-Glycine Gels may result in inaccurate molecular weight estimation.

Product Qualification The Certificate of Analysis (CofA) provides detailed quality control information for each product. The CofA is available on our website at www.lifetechnologies.com/support, and is searchable by product lot number, which is printed on each box.

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HiMark™ Molecular Weight Calculator A molecular weight calculator is available at www.lifetechnologies.com. From the home page, go to Products & Services Controls > Standards, & Ladders > Protein Standards & Ladders > Unstained Protein Standards. Select HiMark™ Standard and follow instructions on the page to download the calculator.

The calculator allows you to easily and accurately calculate the molecular weight of your proteins on NuPAGE® Novex 3–8% and 7% Tris-Acetate Gels and to extrapolate the molecular weight of proteins beyond the standard curve.

Example HiMark™ Unstained HMW Protein Standard (5 μL) was analyzed on a NuPAGE® Novex 3–8% Tris-Acetate Gel (A) or a NuPAGE® Novex 7% Tris-Acetate Gel (B) and stained with SimplyBlue™ SafeStain. The apparent molecular weights of protein standard bands are shown below.

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Accessory Products The following products are available separately and may be used with the HiMark™ Unstained HMW Protein Standard.

Quantity Cat. no. Product

NuPAGE® Novex 3–8% Tris-Acetate Gels 1 box EA0375BOX

NuPAGE® Novex 7% Tris-Acetate Gels 1 box EA0355BOX

NuPAGE® Tris-Acetate SDS Running Buffer (20X)

500 mL LA0041

NuPAGE® LDS Sample Buffer (4X) 10 mL NP0007

SimplyBlue™ SafeStain 1 L LC6060

SilverQuest™ Silver Staining Kit 1 kit LC6070

Limited Use Label License: Research Use Only The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser’s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

©2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

For support visit www.lifetechnologies.com/support or email techsupport@lifetech.com

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