Choose a Molecular Weight Marker Wisely

“Positive and negative controls, as well as molecular size markers, should be included on each gel and blot—either in the main figure or an expanded data supplementary figure”[1] 

After antibody specificity is validated and there is no off-target binding, you can be confident that the right target is detected on your blot. Molecular weight markers or protein ladders are useful in estimating the size of proteins that have already been resolved by gel electrophoresis. They also aid in orienting the gel or membrane quickly and monitoring gel migration. 

Factors to Consider

There are several aspects that can influence the performance of your protein ladder. Keep the following in mind when designing your experiment:

• Depending on the type of buffer, molecular weight markers can run differently.
• Gel type may affect the range of molecular weight markers.
• The molecular weight range of your proteins of interest determines which type of molecular weight marker to use. For example, if you are targeting proteins with a wide range of molecular weights, you should use a marker that reflects that range.
• Molecular weight markers should not be heated. They can, however, be diluted to the same volume as other wells. Because molecular weight markers can be sensitive to agents (e.g., reducing agents or sample buffer), always refer to your data sheet to avoid complications.

Additionally, there are many different types of molecular weight markers. The nature of your experiment can help decide which one is most appropriate.

One-Color Near-Infrared Fluorescence

Odyssey^® One-Color Protein Molecular Weight Marker

For near-infrared fluorescence and one-color detection, utilize the Odyssey One-Color Protein Molecular Weight Marker. It can be used for the 700 nm channel, and its ten protein bands range from 10 to 250 kDa. Additionally, there are reference bands on the ladder at 25, 50, and 75 kDa that are easily visible and three times the intensity of normal bands.

Chameleon^® 700 Pre-Stained Protein Ladder

The Chameleon 700 Pre-stained Protein Ladder is applicable for visible and near-infrared fluorescence in the 700 nm channel. Its seven protein bands range from 8 to 260 kDa and sizes your proteins with multicolored, pre-stained bands.

Chameleon 800 Pre-Stained Protein Ladder

Similarly, the Chameleon 800 Pre-stained Protein Ladder can be used for both visible and near-infrared fluorescence and has six proteins that also range from 8 to 260 kDa. Use this ladder for the 800 nm channel.

Two-Color Near-Infrared Fluorescence

Chameleon Duo Pre-Stained Protein Ladder

For both visual and two-color near-infrared detection, you can use the Chameleon Duo Pre-stained Protein Ladder. It includes eleven different protein bands that range from 8 to 260 kDa.

Chameleon Kit Pre-stained Protein Ladders

For both visual and two-color near-infrared detection, you can use the Chameleon Kit to create your own customized protein ladder. It includes concentrated solutions of Chameleon 700 and Chameleon 800.

Enhanced Chemiluminescence

WesternSure^® Pre-Stained Chemiluminescent Protein Ladder

The WesternSure Pre-stained Chemiluminescent Protein Ladder is applicable to both film and digital detection. This pre-stained, multicolored molecular weight marker does not require strep-tagged HRP or secondary antibody binding. This ladder works with a variety of chemiluminescent substrates available, including our WesternSure PREMIUM Chemiluminescent Substrate.

Protein Ladder Detection

* Chameleon Vue can be detected with chemiluminescence when used with the WesternSure Pen, P/N 926-91000.

Looking for more information? Read up on molecular weight markers and how to choose the best one for your experiment on the “Protein Ladders” info page.

Reference:

1. Image Integrity. Nature. Macmillan Publishers Limited, 2016. Web. 31 July 2017.

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