This page introduces cytoskeletal actin methods for observation and analysis in cell biology.
Page Links: Introduction | Pubmed | External Databases | Swiss Protein | Probes for F-Actin | Probes for G-Actin | Actin Antibodies | Actin Fusion Protein |
Microfilaments consist of actin and tropomyosin in association with a large number of other proteins. These are the smallest (in diameter) of the 3 cytoskeletal filaments that are found in all cells (microfilaments, intermediate filaments, microtubules). Mammalian actin exists in 6 different forms (isoforms) 4 associated with different types of muscle and 2 cytoskeletal isoforms found in all non-muscle cells.
Cytoskeletal actin is generally the most abundant protein found in non-muscle cells and can be identified as a strong 43 Kd band on cell protein extract gels (SDS PAGE).
Actin can be also be identified in its monomer (g-actin, globular) or polymer form (f-actin, filamentous) by specific probes.
Actin can also be identified by a number of commercial and research lab developed antibodies.
Search PubMed Now- Actin Reviews
This image is from the Swiss Protein Database showing monomeric actin bound to DNase1. It also shows sites of ATP binding and calcium.
There are a variety of methods for identifying both polymeric filamentous (F) and monomeric globular (G) actin.
Probes for F-Actin | Probes for G-Actin
Actin Microfilaments can be specifically labelled using probes available from Molecular Probes.
See the special section on microfilaments/A> in their online catalogue and useful table of all their actin probes.
|
Cat # |
Actin-Selective Probe |
Ex/Em (nm) |
|
Coumarin phallacidin |
355/443 |
|
|
NBD phallacidin |
465/536 |
|
|
Alexa 488 phalloidin |
495/518 |
|
|
Fluorescein phalloidin |
496/516 |
|
|
Oregon Green 488 phalloidin |
496/520 |
|
|
BODIPY FL phallacidin |
505/512 |
|
|
Oregon Green 514 phalloidin |
511/528 |
|
|
Eosin phalloidin |
524/544 |
|
|
BODIPY R6G phalloidin |
529/547 |
|
|
Rhodamine phalloidin |
554/573 |
|
|
BODIPY 558/568 phalloidin |
558/569 |
|
|
Alexa 568 phalloidin |
578/600 |
|
|
Alexa 594 phalloidin |
580/609 |
|
|
BODIPY 581/591 phalloidin |
584/592 |
|
|
BODIPY TR-X phallacidin |
589/617 |
|
|
Texas Red-X phalloidin |
591/608 |
|
|
Biotin-XX phalloidin |
NA |
|
|
Phalloidin |
NA |
|
|
Jasplakinolide |
NA |
|
|
Spectra of phallotoxins are either in aqueous
buffer, pH 7-9 (denoted |
||
Rhodamine phalloidin's fluorescence increases upon binding to actin, a phenomenon that allowed Molecular Probes to determine the ligand's binding constant. The binding constants of the other conjugates were determined by competitive binding with rhodamine phalloidin, as described in J Biol Chem 269, 14869 (1994) and Anal Biochem 200, 199 (1992). All binding constants were determined on rabbit skeletal muscle actin. ND = not determined. NA = not applicable.
|
Cat # |
Actin-Selective Probe |
Ex/Em (nm) |
|
DNase I, fluorescein |
494/517 |
|
|
Alexa 488 |
495/519 |
|
|
DNase I, Oregon Green 488 |
496/516 |
|
|
DNase I, tetramethylrhodamine |
555/580 |
|
|
Alexa 594 |
590/617 |
|
|
DNase I, Texas Red |
597/618 |
|
BD Biosciences- Clontech
Clontech have a number of different actin fusion protein plasmids which can be transfected into cells to observe actin protein expression, incorporation and dynamics in living cells.
pEGFP-Actin encoding a fusion protein consisting of the red-shifted, human codon-optimized variant of green fluorescent protein (EGFP) and the gene encoding human cytoplasmic b-actin.
pEYFP-Actin encoding a fusion of the enhanced yellow fluorescent protein (EYFP) and human cytoplasmic b-actin. EYFP is an enhanced yellow-green variant of the Aequorea victoria green fluorescent protein.
pAcGFP1-Actin Vector encoding a green fluorescent protein (GFP) from Aequorea coerulescens (Excitation maximum = 475 nm; emission maximum = 505 nm) and the gene encoding human cytoplasmic b-actin.
Search all NCBI Books for the term "actin"
Search MBoC4 for the term "actin"
These commercial links are for educational purposes only and do not reflect endorsement of a specific product.
BD Biosciences- Clontech Website
This page introduces actin as a cytoskeletal protein that can be identified by a number of different techniques and states. While not all of the above techniques may be used routinely in the Lab, a broad understanding of the use and theoretical background of each technique is essential.
Please email Dr Mark Hill if you wish to make a comment about this current project.
