Fluorescent Cell Imaging Simplified

Video Tutorial: Perfect Your Quantitative Western Blotting

Are you getting the most from your western blot data? Does Imaging technique matter? Film or Imager? In this video, we describe a methodology to obtain reliable quantitative data from chemiluminescent western blots using standardization procedures coupled with the updated reagents and detection methods. For the best resolution, watch the video in full screen at HD resolution.

Which camera is best for chemiluminescent detection?

As illustrated in the chart below, there are numerous methods for documenting your western blotting results. In this post we will review the use of charged couple display (CCD) cameras for chemiluminescence detection of western blots. The information is reprinted from the Protein Blotting Guide from Bio-Rad Laboratories. Click on the picture to enlarge the chart.

Charged couple display (CCD) imaging is the easiest, most accurate, and rapid method for chemiluminescent detection. CCD cameras are versatile systems that image both gels and blots, and operate with either trans-illumination provided by light boxes (visible or UV) positioned underneath the gel for imaging a variety of stains (Coomassie, silver, fluorescence) or epi-illumination of blots detected using colorimetric or fluorescence techniques. Different illumination wavelengths are available for multiplex fluorescence immunodetection. CCD cameras can also be used without illumination to detect luminescent signals. Supercooled CCD cameras reduce image noise, allowing detection of faint luminescent signals.

CCD cameras have a linear response over a broad dynamic range — 2–5 orders of magnitude — depending on the bit depth of the system. CCD cameras also offer convenience by providing a digital record of experiments for data analysis, sharing, and archiving, and by eliminating the need to continually purchase consumables for film development. CCD cameras also approach the limit of signal detection in a relatively short time. For example, the VersaDoc™ MP 5000 imaging system can reach the limit of detection of a given experiment in <1 min, compared to 30 min required by Kodak Bio-Max film for the same experiment.

For more information on Bio-Rad’s line of imaging systems visit www.bio-rad.com/imaging.

Video Tutorial: Analyzing Images with Image Lab Software

In a previous video tutorial, we showed you how to acquire an image using Image Lab software from Bio-Rad Laboratories. In this video, you will learn how to use the software to analyze images. Please note that in addition to analyzing images acquired by the program itself, Image Lab software can also analyze images captured by other programs such as Quantity One. Also, the software is not licence protected and can be installed on any computer in your lab.

For ideal viewing, please maximize the video window by clicking on the full screen button in the bottom right hand corner of the video screen.

Don’t be manipulated by your imaging data

Have you ever heard the term “ignorance is bliss?” Not in science. For scientists, ignorance is death! Yet many of us rely on the accuracy of our lab equipment without actually knowing how they work. For example, did you know that not all imaging systems provide you with accurate data?

Scientific data collected with an imaging device can be true or altered. Alteration of imaged data is used to create an impression of more data retrieved than is actually collected.

Don’t be fooled by your imaging data! Read Not all bits are created equal to understand more.