Immunofluorescence analysis of Cryptochrome 1 was performed using 70% confluent log phase U-87 MG 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 Cryptochrome 1 Rabbit Polyclonal Antibody (PA1-527) at 2µg/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 conjµgate (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||Human|
|Published species reactivity||Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic Peptide: C Q(594) S V G P K V Q R Q S S N(606)|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid 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|
|Western Blot (WB)||1:2,000|
* 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.
|Immunocytochemistry (ICC)||See 1 publications below|
PA1-527 detects recombinant human cryptochrome 1 (Cry1).
PA1-527 has been successfully used in Western blot procedures. By Western blot, this antibody detects an ~70 kDa protein representing full length recombinant human Cry1 protein.
PA1-527 immunizing peptide corresponds to amino acid residues 594-606 from mouse Cry1. PA1-527 immunizing peptide (Cat. # PEP-103) is available for use in neutralization and control procedures.
Cryptochromes (Cry1 and 2) are blue, ultraviolet-A photoreceptor pigment proteins that are involved circadian rhythm regulation in plants and animals. In mammals, Cry1 and 2 expression oscillates with respect to the daily light-dark cycle in the suprachiasmatic nucleus of the hypothalamus. These proteins localize to the cell nucleus, interact with each of the Per proteins, and assist in the translocation of Per from the cytoplasm to nucleus.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice.
PA1-527 was used in immunocytochemistry to assay for Cry1 in eyes of the Opn4-/- Gnat1-/- Cnga3-/- mice
|Hattar S,Lucas RJ,Mrosovsky N,Thompson S,Douglas RH,Hankins MW,Lem J,Biel M,Hofmann F,Foster RG,Yau KW||Nature (424:76)||2003|