Have researchers visualized LC3-puncta by fluorescence microscopy, where, morphologically, the puncta accumulate in a pattern similar to a ‘string of pearls’ one after another after another in long string that slowly curve and occasionally branch from one-another?
Hi,
Can anybody suggest me good anti mouse Atg5 and anti mouse GFAP antibodies for Immunofluorescence assays?
Thanks in advance.
Veeru Patil
It is well established that GFP fluorescence is quenched in the acidic environment of the lysosome. However, does anyone know if this is the case as well in fixed cells? Would the fixation method influence this (methanol vs formaldehyde)?
Thank you
Company: all companies
Catalog #: check it
Comments:
It has come to our attention that the price of Bafilomycin can fluctuate within one order of magnitude, with some of the better known companies being on the most expensive side. Worth checking around when ordering.
Posted by: Nicholas T. Ktistakis
Institution: Signalling Programme; Babraham Institute; Babraham, Cambridge UK
Company: Novus Biologicals, Abgent
Catalog #: NB100-2220SS, AP1800a, AP1801b, AP1803a
Comments:
These antibodies to human LC3 do not show cross-reactivity for planarian LC3 (C. González-Estévez, personal communication).
Posted by: Daniel J. Klionsky
Institution: Life Sciences Institute and Department of Molecular, Cellular and Developmental Biology, and Department of Biological Chemistry; University of Michigan; Ann Arbor, MI USA
Company: Addgene
Catalog #: 21074
Comments:
The tandem mRFP-EGFP-LC3 plasmid (ptfLC3) described by Tamotsu Yoshimori (Kimura et al. Autophagy 2007; 3:452-60) is available from the nonprofit company Addgene. This construct can be used to follow autophagic flux in mammalian cells.
Posted by: Daniel J. Klionsky
Institution: Life Sciences Institute and Department of Molecular, Cellular and Developmental Biology, and Department of Biological Chemistry; University of Michigan; Ann Arbor, MI USA
Company: Abgent
Catalog #: AP3301a
Comments:
The antibody to LC3 phosphorylated at S12 recognizes rat LC3 as well as LC3 in human cell lines and mouse primary neurons, when used at 1/500 for western blot. Phosphorylation is decreased upon autophagy induction.
Reference: http://jcb.rupress.org/content/early/2010/08/11/jcb.201002108.full
Cherra SJ, 3rd, Kulich SM, Uechi G, Balasubramani M, Mountzouris J, Day BW, Chu CT: Regulation of the autophagy protein LC3 by phosphorylation, J Cell Biol 190:533-539
In primary neurons, the band co-migrates with LC3-I. In tumor cell lines, there were additional slightly higher molecular weight bands, and in some tumor cells the ~20 kDa band predominates. An antibody to a C-terminal region of LC3 (AP1802b, 1/500) recognizes the band(s) recognized by phospho-LC3. It is unclear whether the phosphorylation affects affinity for N-terminal antibodies for LC3.
As the Journal of Cell Biology editorial policy is to remove catalog numbers from Methods, I have appended a table of antibodies used.
Antibody Cat # Company Dilution Assay
Cleaved-LC3 AP1806a Abgent 1:250 IF
LC3 Nterm (2G6) 0260-100Nanotools …1:500 WB, cell lines
…1:1000 or more WB, 1° neurons
LC3 Cterm AP1802b Abgent 1:500 WB
pLC3 S12 AP3301a Abgent 1:500 WB
pLC3 T12 AP3530a Abgent 1:500 WB
GFP A6455 Invitrogen 1:5000 IF & WB
HA (16B12) MMS-101R Covance 1:1000 IF
GAPDH Ab37168 Abcam 1:10,000 WB
Lamin A/C 2032 Cell signalling 1:1000 WB
β-Actin A5316 Sigma 1:5000 WB
Atg7600-401-487 Rockland 1:1000 WB
Posted by: Charleen T. Chu
Institution: University of Pittsburgh School of Medicine
We would like to examine the mouse p62 and LC3 mRNA level in MEF cells. Could someone kindly provide us the workable primers and the according molecular weight? Your generous support will be deeply appreciated!