Invitrogen

CLOCK Polyclonal Antibody

Product Image Gallery

CLOCK Antibody — Published figure using CLOCK polyclonal antibody (Product # PA1-520)

CLOCK Antibody in Immunohistochemistry (Paraffin) (IHC (P)) — Immunohistochemistry analysis of CLOCK showing positive staining in the nucleus and cytoplasm of paraffin-treated Human colon tissue (right) compared with a negative control in the absence of primary antibody (left). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a CLOCK polyclonal antibody (Product # PA1-520) diluted by 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

CLOCK Antibody in Immunofluorescence (IF) — Immunofluorescent analysis of CLOCK (green) showing staining in the nucleus and cytoplasm of U251 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a CLOCK polyclonal antibody (Product # PA1-520) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 °C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

CLOCK Antibody in Western Blot (WB) — Western blot analysis of CLOCK was performed by loading 25 µg of Hela (lane 1) and NIH-3T3 (lane 2) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4°C overnight. The membrane was probed with a CLOCK polyclonal antibody (Product # PA1-520) at a dilution of 1:4000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~100 kDa.

Product Details

Tested Applications

Dilution

Immunocytochemistry (ICC)

1:10-1:200

Immunofluorescence (IF)

1:10-1:200

Immunohistochemistry (Frozen) (IHC (F))

1:100-1:1000

Immunohistochemistry (Paraffin) (IHC (P))

1:100-1:1000

Western Blot (WB)

1:200

Published Applications

Western Blot (WB)

See 2 publications below

Immunohistochemistry - Free Floating (IHC (Free))

See 1 publication below

Gel Shift (GS)

See 1 publication below

Immunohistochemistry (IHC)

See 3 publications below

Product Specifications

Species Reactivity

Hamster, Human, Mouse, Rat

Published species

Human, Mouse

Host / Isotype

Rabbit / IgG

Class

Polyclonal

Type

Antibody

Immunogen

Synthetic Peptide: S(11) S I V D R D D S S I F D G L V E E D D(30) C

Conjugate

Unconjugated

Form

Liquid

Concentration

1 mg/mL

Purification

Antigen affinity chromatography

Storage buffer

PBS with 1mg/mL BSA

Contains

0.05% sodium azide

Storage conditions

-20° C, Avoid Freeze/Thaw Cycles

RRID

AB_2082586

Product Specific Information

PA1-520 detects CLOCK from human, mouse, rat and hamster tissues.

PA1-520 has been successfully used in Western blot, ICC/IF and immunohistochemistry procedures. By Western blot, this antibody detects an ~100 kDa protein representing CLOCK from rat brain homogenate. Immunohistochemical staining of CLOCK in hamster brain with PA1-520 results in the staining of the superchiasmatic nucleus.

PA1-520 immunizing peptide corresponds to amino acid residues 11-30 of mouse CLOCK. This sequence is completely conserved between mouse and human CLOCK. PA1-520 immunizing peptide (Cat. # PEP-072) is available for use in neutralization and control experiments.

Target Information

Circadian rhythmicity is a basic property of phylogenetically diverse organisms which range from animals and plants, to fungi. Regulation of endogenous biological clocks is regulated at the genetic level by a protein-mediated, autoregulatory feed-back loop. In mammals, several genes that encode members of the basic helix-loop helix (bHLH) PAS (PER-ARNT-SIM) transcription factor family have been shown to play a significant role in regulating circadian oscillations. Transactivation of CLOCK-induced genes is mediated via an E box enhancer (CACGTG) found upstream of target genes. CLOCK-ARNT3 heterodimers bind to E box regulatory elements and stimulate gene transcription. CLOCK has been shown to transactivate the mammalian homolog of Drosophila per. PER, in concert with the product of the mammalian timeless gene (TIM), negatively regulates its own transcription by blocking the activity of the CLOCK-BMAL1 transactivation complex.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: bHLHe8; circadian locomoter output cycles kaput; circadian locomoter output cycles kaput protein; Circadian locomoter output cycles protein kaput; Circadium Locomotor Output Cycles Kaput; Class E basic helix-loop-helix protein 8; clock circadian regulator; clock homolog; hCLOCK; mCLOCK; rCLOCK

Gene Aliases: 5330400M04Rik; BHLHE8; CLOCK; I79_020252; KAT13D; KIAA0334

UniProt ID: (Human) O15516, (Mouse) O08785, (Rat) Q9WVS9

Entrez Gene ID: (Human) 9575, (Chinese hamster) 100770092, (Mouse) 12753, (Rat) 60447

Molecular Function: basic helix-loop-helix transcription factor transcription factor