Immunofluorescence analysis of ATG5 was performed using 70% confluent log phase PC-3 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ATG5 Rabbit Polyclonal Antibody (PA146178) at 2ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification
|Tested species reactivity||Bovine, Human, Mouse, Non-human primate, Pig, Rat, Xenopus, Zebrafish|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide made to an N-terminal region of the human ATG5 protein.|
|Purification||Antigen affinity chromatography|
|Contains||0.02% sodium azide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Immunohistochemistry (Paraffin) (IHC (P))||1:400|
|Western Blot (WB)||2 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
In Western Blot, a band is seen ~56 kDa representing the ATG5-ATG12 complex, the molecular weight of human ATG5 is ~33 kDa.
Prior to immunostaining paraffin tissues, antigen retrieval with sodium citrate buffer (pH 6.0) is recommended.
Suggested positive control: antigen standard for ATG5 (transient overexpression lysate), mouse wildtype ES cell lysate.
Autophagy is an important mechanism for nonselective intracellular breakdown whereby cytosol and organelles are encapsulated in vesicles, which are then engulfed and digested by lytic vacuoles/lysosomes. ATG5 is required for autophagy. It conjugates to ATG12 and associates with isolation membrane to form autophagosomes. The conjugate detaches from the membrane immediate before or after autophagosomes formation is completed. Conjugation to ATG12 is essential to autophagy, but is not required for association with isolation membrane. ATG5 also plays an important role in the Apoptotic process. It's expression is a relitively late event in the apoptotic process, occuring down stream of caspase activity.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.