Imaging

How to get the best results for fluorescent western blots

Fluorescence is typically more sensitive than chemiluminescence, but there are still some pitfalls to avoid.

How to merge images so that you can view the chemiluminescent blot and the visual stained ladder at the same time:

In many cases it is very useful to be able to view the ladder bands and the chemiluminescent signal from your blot at the same time. Using Quantity One software and the Versadoc imaging system here is an example.

1. You must first take a picture of the blot in chemi mode. Without moving the blot, you need to take a picture with the white lights on in visual mode.

2. To take the picture with the white lights, select ''channel 2'' from the acquisition screen and click on enable checkbox.

 

Scientific Gel and Blot Imaging: The difference between resolution and sensitivity with CCD cameras

What is more important? Resolution or sensitivity? The answer depends on your application. If for example you are running 2-D gels or wish to visualize microarrays, then resolution is by far more important than sensitivity. If on the other hand, you are trying to visualize a very weak signal like a chemiluminescent western blot, or a fluorescent probe bound to a protein with very low expression, then sensitivity is far more important.

Comparison of LiCor system to Versadoc

There was a time when to use fluorescent probes for a western blot the major problem was that the membrane itself would autofluoresce. All of that has changed due to the introduction of low fluorescent membranes which can be purchased from Millipore (Immobilon-FL PVDF Cat no. IPFL000W0) or Pall (Cat no. PVM020C-099)

LiCor got around the problem by using red-shifted excitation sources which did not light up the membrane but would still excite Alexa Dyes. Now you can use the Versadoc imaging system which is not limited to using red dyes to image Alexa-488, 555, 633, 647

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