Fluorescent Secondary Antibodies from Li-Cor Simplify Western Blot Quantification

March 29, 2016

Medical Physics
University of Wisconsin
Postdoctoral Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

Li-Cor Technologies

Product Name:

IRDye 800CW Donkey anti-Mouse IgG

Catalog Number:

926-32212

We examine protein expression by Western blot analysis. By using multiplexing techniques, we can quantify the amount of our protein of interest in cell lines. In this study, we evaluated the expression of CD146 in three human lung cancer cell lines. We chose the LiCor secondary antibody because it works well with the LiCor imaging platform.

Experimental Design and Results Summary

Applications

Western Blot

Sample

Three human lung cancer cell lines

Primary Incubation

Mouse-derived monoclonal CD146 antibody (1:100; Santa Cruz) and rabbit β-actin (1:400; LI-COR Biosciences) incubated in iBind system (25 degree Celcius)

Blocking Agent

iBind Fluorescent Detection Solution Kit (pre-made blocking agent)

Secondary Incubation

Donkey anti-rabbit IRDye 680RD and donkey anti-mouse 800CW (1:15,000) at 25 degree Celcius in iBind system

Tertiary Incubation

There was no tertiary incubation. Western blot was removed from iBind system for imaging.

Detection

Flourescence detection with LiCor Odyssey system

Results Summary

Two bands were seen in each Western blot at ~110 kDa corresponding to CD146 and β-actin was visible at 38 kDa. The band for H460 cell line displayed the highest signal, indicative of the highest CD146 expression. The other cell lines showed varying levels of CD146 expression.

Additional Notes

None

Image Gallery

Summary

The Good

Excellent sensitivity, simple to use.

The Bad

Requires optimization (as too much will cause imaging problems).

The Bottom Line

IRDye 800CW Donkey-anti Mouse IgG is a great secondary antibody for Western blots, especially for quantitative Western blotting procedures. Make sure to use the correct amount of antibody (less than 1 microliter), as too much antibody will completely over-stain the membrane.

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