Beta-galactosidase: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
(links to protocols and X-Gal) |
||
(10 intermediate revisions by 2 users not shown) | |||
Line 11: | Line 11: | ||
*You can measure lacZ activity using flow cytometry. See [http://www.cyto.purdue.edu/flowcyt/research/micrflow/jepras/jepras2.htm A flow cytometric study of stationary phase gene expression in E.coli using lacZ reporter gene fusion] | *You can measure lacZ activity using flow cytometry. See [http://www.cyto.purdue.edu/flowcyt/research/micrflow/jepras/jepras2.htm A flow cytometric study of stationary phase gene expression in E.coli using lacZ reporter gene fusion] | ||
**This paper uses [http://probes.invitrogen.com/media/pis/mp02904.pdf C<sub>12</sub>FDG] from Invitrogen. However, FDG might be a better substrate in gram-negative bacteria like ''Escherichia coli'' <cite>Plovins-ApplEnvironMicrobio-1994</cite> | **This paper uses [http://probes.invitrogen.com/media/pis/mp02904.pdf C<sub>12</sub>FDG] from Invitrogen. However, FDG might be a better substrate in gram-negative bacteria like ''Escherichia coli'' <cite>Plovins-ApplEnvironMicrobio-1994</cite> | ||
* Another fluorogenic substrate is 4-methylumbelliferyl β-D-galactopyranoside (MUG) which works in bacteria, yeast, and mammalian cells (without requiring permeabilization/lysis). <cite>Vidal-Aroca-2006</cite> | |||
*Alpha-complementation of β-galactosidase does not seem to yield activities equal to wildtype β-galactosidase. Depending on the fragment, the activity can be up to 24% of wildtype <cite>Zamenhof-JBacteriol-1972</cite>. '''(If anyone has a better reference comparing results from a Miller assay of alpha-complementated β-galactosidase with wildtype, please include it here.)''' | |||
==Protocols== | |||
* [[beta-galactosidase assay]] | |||
* [[LacZ staining of cells]] | |||
==See also== | |||
* [[X-Gal]] | |||
==Reference== | ==Reference== | ||
Line 18: | Line 27: | ||
#Ullmann-Bioessays-1992 pmid=1345751 | #Ullmann-Bioessays-1992 pmid=1345751 | ||
#Plovins-ApplEnvironMicrobio-1994 pmid=7811104 | #Plovins-ApplEnvironMicrobio-1994 pmid=7811104 | ||
#Miller-1972 isbn=0879691069 | |||
// original β-galactosidase assay by Miller | |||
#Zamenhof-JBacteriol-1972 pmid=4552986 | |||
#Thibodeau-Biotechniques-2004 pmid=15038156 | |||
#WorthingtonBiochem http://www.worthington-biochem.com/BG/default.html | |||
#Serebriiskii-AnalBiochem-2000 pmid=10998258 | |||
#Vidal-Aroca-2006 pmid=16629389 | |||
</biblio> | </biblio> | ||
[[Category:Material]] [[Category:Enzyme]] [[Category:Reporter]] | [[Category:Material]] [[Category:Enzyme]] [[Category:Reporter]] |
Latest revision as of 08:28, 10 June 2008
Some interesting facts [1]
- A tetramer of 4 identical subunits
- Each subunit is 120kD.
- Active only as a tetramer.
- Mutations in some of the codons of the N-terminal 60aa or C-terminal 100aa results in an inactive, dimeric β-galactosidase. [2]
- If the above sequences are deleted, the missing protein fragment can be replaced by the corresponding peptide. This is called intracistronic alpha or omega complementation respectively.
- Its N-terminal 23 residues can be replaced by any amino acid residues without affecting the enzymatic activity.
- A mutant with an internal deletion of codons 21-41 of the lacZ gene does not produce any active β-galactosidase.
- A mutant with a deletion of everything past residue 60 (i.e. it expresses only the first 60 N-terminal amino acids) does not produce any active β-galactosidase.
- You can measure lacZ activity using flow cytometry. See A flow cytometric study of stationary phase gene expression in E.coli using lacZ reporter gene fusion
- Another fluorogenic substrate is 4-methylumbelliferyl β-D-galactopyranoside (MUG) which works in bacteria, yeast, and mammalian cells (without requiring permeabilization/lysis). [4]
- Alpha-complementation of β-galactosidase does not seem to yield activities equal to wildtype β-galactosidase. Depending on the fragment, the activity can be up to 24% of wildtype [5]. (If anyone has a better reference comparing results from a Miller assay of alpha-complementated β-galactosidase with wildtype, please include it here.)
Protocols
See also
Reference
- ISBN:3-11-014830-7
- ISBN:0317118099
- Plovins A, Alvarez AM, Ibañez M, Molina M, and Nombela C. Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells. Appl Environ Microbiol. 1994 Dec;60(12):4638-41. DOI:10.1128/aem.60.12.4638-4641.1994 |
- Vidal-Aroca F, Giannattasio M, Brunelli E, Vezzoli A, Plevani P, Muzi-Falconi M, and Bertoni G. One-step high-throughput assay for quantitative detection of beta-galactosidase activity in intact gram-negative bacteria, yeast, and mammalian cells. Biotechniques. 2006 Apr;40(4):433-4, 436, 438 passim. DOI:10.2144/000112145 |
- Zamenhof PJ and Villarejo M. Construction and properties of Escherichia coli strains exhibiting -complementation of -galactosidase fragments in vivo. J Bacteriol. 1972 Apr;110(1):171-8. DOI:10.1128/jb.110.1.171-178.1972 |
- Ullmann A. Complementation in beta-galactosidase: from protein structure to genetic engineering. Bioessays. 1992 Mar;14(3):201-5. DOI:10.1002/bies.950140311 |
- ISBN:0879691069
original β-galactosidase assay by Miller
- Thibodeau SA, Fang R, and Joung JK. High-throughput beta-galactosidase assay for bacterial cell-based reporter systems. Biotechniques. 2004 Mar;36(3):410-5. DOI:10.2144/04363BM07 |
- Serebriiskii IG and Golemis EA. Uses of lacZ to study gene function: evaluation of beta-galactosidase assays employed in the yeast two-hybrid system. Anal Biochem. 2000 Oct 1;285(1):1-15. DOI:10.1006/abio.2000.4672 |