Chemiluminescence Detection. Using UVP s ChemiDoc-It System
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1 Chemiluminescence Detection Using UVP s ChemiDoc-It System
2 Protein Detection Agents Chemiluminescence Fluorescence Colorimetric Detection Radioisotopic Detection
3 Chemiluminescence Generation of light through sample s own light source by chemical reaction, without involvement of heat Chemiluminescence is widely used for protein blot detection Bioluminescence is chemiluminescence that takes place inside a living organism For purposes of limiting the discussion on chemiluminescence applications using the chemi doc-it, we will only cover chemiluminescent imaging of protein blots Its basic strength is its simplicity as a detection tool virtually no background or autofluorescence of samples to worry about; offers the same sensitivity as film, is less expensive than film and offers the speed which is a big drawback for film.
4 Fluorescence Absorption and emission of light using light energy When used in protein detection, it involves the use of fluorescent stains and transilluminated or epi illuminated light sources Just like chemiluminescence, fluorescence can be used for detecting signal inside a living organism (see ibox) Basic strength is the simplicity and speed of imaging and the richness of color; major weakness is the high background or autofluorescence of samples that can be addressed by proper filtering.
5 Colorimetric Detection Colorimetric or visible light imaging (from 480 nm to about 700 nm) using white light Generally makes use of stains such as coomassie blue and silver stain in imaging protein samples as Elisa/Blots. Uses white light to illuminate the samples
6 Radioisotopic Imaging Use of radioisotopes (32p, 35s etc.) End result is usually an X-Ray film which is then read using white light illumination or a scanner Safety issues, cost and speed are typically its downside
7 Chemiluminescence Applications For Proteins & DNA/RNA Western Blots (proteins) Dot/Slot Blots (proteins & DNA/RNA) Northern Blots (RNA) Southern Blots (DNA) Western Blot Southern Blot Dot Blot
8 Chemiluminescent Substrates Commonly Used Substrates: ECL Pierce Rockland FemtoMax(recommended substrate with femtogram level sensitivity) Compared with radioisotopes, chemi substrates will have the same sensitivity (down to femtogram level) & faster detection; these do not have any health and hazardous issues associated with the useand disposal of radioisotopes.
9 Chemiluminescent CCD Imaging Deeply cooled cameras with same sensitivity, higher dynamic range, higher linearity than film are currently available Super light-tight darkroom, necessary for low light imaging should also be a part of the system Capture and Analysis software to perform sequential images at same or varying exposure times as well as powerful analysis features should likewise be part of the imaging system
10 Chemiluminescence Detection Using UVP s ChemiDoc-It System Deeply Cooled, 2.0 megapixel monochrome Chemi 410 camera with highly sensitive optics & high quantum efficiency Light tight, super dark and compact darkroom Adjustable chemi tray for varying heights to obtain desired field of view VisionWorks Software For acquisition and analysis of chemi images
11 VisionWorks LS Software
12 Image Capture Straightforward Capabilities
13 Image Capture Sequential Capture of Chemi Western Blot
14 Imaging Tools Using Invert & Histogram Tools to Optimize Chemi Western Blots
15 Imaging Tools Using Invert & Histogram Tools to Optimize Chemi Western Blots
16 Imaging Tools Recording & Saving Macros
17 Analysis Tools Intuitive, Easy to Use Analysis Tools Area Density Band Density and MW Calculations Scan Lanes for Purity
18 Area Density For Measuring Intensity
19 ID Analysis For Band Density & Molecular Weight Calculations
20 1D Analysis To Scan Lane for Purity
21 Analysis Results Exportable to Excel
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