- Coomassie Stains & Protein Gel Documentation
- Nucleic Acid Gel Documentation
- ELISA
- EMSA/Gel Shift Assay
- Glycoprotein Detection
- In-Cell Western™ Assay
- In-Gel Western Assay
- Microwestern Array
- Northern Blot
- On-Cell Western Assay
- Protease Assay
- Protein Array
- Quantitative Western Blot
- Reporter Gene Assay
- Reverse Phase (Lysate) Array
- RNAi Analysis
- Small Animal Imaging
- Southern Blot
- Tissue Section Imaging
- Transcription Factor Assay
Email bio-eu@licor.com
APPLICATIONS for the
ODYSSEY® CLx
Applications for the Odyssey CLx Infrared Imaging System
IN-GEL WESTERN ASSAY
Application Overview
Figure 1
In-Gel Western detection avoids transfer problems by directly detecting target proteins within the polyacrylamide gel matrix, using the Odyssey® CLx Infrared Imaging System. Advantages of infrared in-gel detection include:
Elimination of transfer steps and membranes – reducing time and cost
Clean images, minimal background banding
Nanogram to picogram detection sensitivity
Multiplexed, two-color detection
Clearer, sharper bands than in-gel chemiluminescent protocols
Wide linear range
In-Gel Western detection is ideal for hard-to-transfer proteins, including
Large, hydrophobic, or post-translationally modified proteins
Small proteins that may migrate through the blotting membrane and be lost
Proteins that are not well-retained by the membrane1
[ABOVE] Figure 2. Beginning with 10 ng/lane, two-fold serial dilutions of purified human Transferrin were subjected to electrophoresis followed by detection using an in-gel Western Protocol. Reprinted with permission from Urh, M et al. Poster presentation, Advances in Genome Biology and Technology Conference (2002)
[LEFT] Figure 3.
In-gel detection of Cytochrome P450 3A4 (CYP3A4) Fixed gel was probed with anti-CYP3A4 primary antibody and IRDye800 secondary antibody. The limit of detection is approximately 3 ng. Reprinted with permission from Theisen, MJ and Chiu, ML.
LI-COR Biosciences (2004)
1 Theisen, MJ and Chiu, ML. In-gel immunochemical detection of proteins that transfer poorly to membranes. LI-COR Biosciences (2004)
Technical Resources
Application Notes
For related protocols, see Western blotting
White Paper
Theisen, MJ and Chiu, ML.
In-gel immunochemical detection of proteins that transfer poorly to membranes
Poster
Urh, M et al.
The Use of IR Fluorescent Technology for the Analysis of Signal Transduction and Protein-Nucleic Acid Interactions
Poster presentation, Advances in Genome Biology and Technology Conference (2002)
Webinars
“Odyssey Infrared Imaging System” video tutorial by Amy Geschwender
For related video tutorials, see Western blotting
PubMed
Desplats C et al.
Characterization of Nda2, a plastoquinone-reducing type II NAD(P)H dehydrogenase in chlamydomonas chloroplasts.
J Biol Chem. 284(7):4148-57(2009)Itatsu, K et al.
Cyclooxygenase-2 is involved in the up-regulation of matrix metalloproteinase-9 in cholangiocarcinoma induced by tumor necrosis factor-alpha.
Am J Pathol. 174(3):829-41(2009)Gao, X et al.
Near-infrared fluorescence detection of ATP-biotin-mediated phosphoprotein labeling.
Biotechnol Lett. 31(1):113-7(2009)Chalupnikova, K et al.
Recruitment of the RNA helicase RHAU to stress granules via a unique RNA-binding domain.
J Biol Chem. 283(50):35186-98(2008)Kumar, V et al.
Enzyme source effects on CYP2C9 kinetics and inhibition.
Drug Metab Dispos. 34(11):1903-8(2006)Morrison CJ and Overal CM.
TIMP independence of matrix metalloproteinase (MMP)-2 activation by membrane type 2 (MT2)-MMP is determined by contributions of both the MT2-MMP catalytic and hemopexin C domains.
J Biol Chem.281(36):26528-39(2006)Pelman GR et al.
Pivotal molecular determinants of peptidic and collagen triple helicase activities reside in the S3' subsite of matrix metalloproteinase 8 (MMP-8): the role of hydrogen bonding potential of ASN188 and TYR189 and the connecting cis bond.
J Biol Chem. 280(3):2370-7(2005)Tam EM et al.
Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin c domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities.
J Biol Chem. 279(41):43336-44(2004)
