Home > Product > Antibody > Rabbit Anti-ATF4 antibody
Cyclic AMP response element binding protein 2; DNA binding protein TAXREB67; Activating Transcription Factor 4; ATF 4; ATF4 protein; CREB 2; CREB2; CREB-2; Cyclic AMP dependent transcription factor ATF 4; Tax Responsive Enhancer Element B67; TAXREB67; TXR
Cat:
SL1531R
Species Reactivity:
Human,Mouse,Rat,(predicted: Dog,Pig,Cow,Horse,Rabbit,Sheep,)
Immunogen:
KLH conjugated synthetic peptide derived from human ATF4:251-351/351
Format:
Liquid
Storage instructions:
Shipped at 4℃. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles.
Concentration:
1mg/ml
Clonality:
Polyclonal
Isotype:
IgG
Applications:
WB=1:500-2000ELISA=1:5000-10000IHC-P=1:100-500IHC-F=1:100-500Flow-Cyt=0.2μg /testICC=1:100IF=1:100-500(Paraffin sections need to do antigen repair)not yet tested in other applications.optimal dilutions/concentrations should be determined by the end user.
Host:
Rabbit
Product Overview:
Sample: Embryo(Mouse) lysate at 30ug;Eye(Mouse) lysate at 30 ug; Primary: Anti-ATF4 (SL1531R) at 1:300 dilution; Secondary: HRP conjugated Goat-Anti-Rabbit IgG(bse-0295G) at 1: 5000 dilution; Predicted band size : 38kDObserved band size : 38kDSample: Lane 1: Mouse Spleen Lysates Lane 2: Mouse Lung Lysates Lane 3: Mouse Brain Lysates Lane 4: Rat Brain Lysates Lane 5: Human Hela cell Lysates Lane 6: Human HepG2 cell Lysates Primary: Anti-ATF4 (SL1531R) at 1/1000 dilutionSecondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilutionPredicted band size: 38kDaObserved band size: 50kDaSample: Cerebrum (Rat) Lysate at 40 ugCerebrum (Mouse) Lysate at 40 ugPrimary: Anti-ATF4 (SL1531R) at 1/1000 dilutionSecondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilutionPredicted band size: 37’50 kDObserved band size: 50 kDParaformaldehyde-fixed, paraffin embedded (human breast carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (human brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (rat spleen); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (rat lung); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (Human breast cancer); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.Paraformaldehyde-fixed, paraffin embedded (human brain glioma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining. Paraformaldehyde-fixed, paraffin embedded (Rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.Paraformaldehyde-fixed, paraffin embedded (Mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.Tissue/cell: human rectal carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min; Incubation: Anti-ATF4 Polyclonal Antibody, Unconjugated(SL1531R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) stainingTissue/cell: human gastric carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min; Incubation: Anti-ATF4/CREB-2 Polyclonal Antibody, Unconjugated(SL1531R) 1:200, overnight at 4℃, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) stainingU2OS cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (ATF4) polyclonal Antibody, Unconjugated (SL1531R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (ATF4) polyclonal Antibody, Unconjugated (SL1531R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.Blank control (blue line): Hela Primary Antibody (green line): Rabbit Anti-Bid antibody (SL1531R) Dilution: 0.2μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITCDilution: 1μg /test. ProtocolThe cells were fixed with 70% ethanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 30 min on ice. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.Blank control:Mouse spleen. Primary Antibody (green line): Rabbit Anti-ATF4 Alpha antibody (SL1531R) Dilution: 2μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-FITCDilution: 1μg /test. ProtocolThe cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
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This gene encodes a transcription factor that was originally identified as a widely expressed mammalian DNA binding protein that could bind a tax-responsive enhancer element in the LTR of HTLSLV1. The encoded protein was also isolated and characterized as the cAMP-response element binding protein 2 (CREB-2). The protein encoded by this gene belongs to a family of DNA-binding proteins that includes the AP-1 family of transcription factors, cAMP-response element binding proteins (CREBs) and CREB-like proteins. These transcription factors share a leucine zipper region that is involved in protein-protein interactions, located SLCterminal to a stretch of basic amino acids that functions as a DNA binding domain. Two alternative transcripts encoding the same protein have been described. Two pseudogenes are located on the X chromosome at q28 in a region containing a large inverted duplication. [provided by RefSeq, Sep 2011]

Function:
Transcriptional activator. Binds the cAMP response element (CRE) (consensus: 5'-GTGACGT[AC][AG]-3'), a sequence present in many viral and cellular promoters. Cooperates with FOXO1 in osteoblasts to regulate glucose homeostasis through suppression of beta-cell production and decrease in insulin production. It binds to a Tax-responsive enhancer element in the long terminal repeat of HTLSLVI. Regulates the induction of DDIT3/CHOP and asparagine synthetase (ASNS) in response to ER stress. In concert with DDIT3/CHOP, activates the transcription of TRIB3 and promotes ER stress-induced neuronal apoptosis by regulating the transcriptional induction of BBC3/PUMA. Activates transcription of SIRT4.

Subunit:
Binds DNA as a homo- or heterodimer. Interacts (via its leucine zipper domain) with GABBR1 and GABBR2 (via their SLCtermini). Interacts (via its DNA binding domain) with FOXO1 (SLCterminal half); the interaction occurs in osteoblasts and regulates glucose homeostasis through suppression of beta-cell proliferation and a decrease in insulin production. Interacts with SATB2; the interaction results in enhanced DNA binding and transactivation by these transcription factors. Interacts with CEP290 (via an N-terminal region). Interacts with NEK6, DAPK2 (isoform 2) and ZIPK/DAPK3. Forms a heterodimer with TXLNG in osteoblasts. Interacts with DDIT3/CHOP.

Subcellular Location:
Cytoplasm. Cell membrane. Nucleus. Cytoplasm, cytoskeleton, microtubule organizing center, centrosome. Note=Colocalizes with GABBR1 in hippocampal neuron dendritic membranes. Co- localizes with NEK6 in the centrosome.

Post-translational modifications:
Ubiquitinated by SCF(BTRC) in response to mTORC1 signal, followed by proteasomal degradation and leading to down-regulate expression of SIRT4.
Phosphorylated by NEK6. Phosphorylated on the betaTrCP degron motif at Ser-219, followed by phosphorylation at Thr-213, Ser-224, Ser-231, Ser-235 and Ser-248, promoting interaction with BTRC and ubiquitination. Phosphorylation is promoted by mTORC1.
Phosphorylated by NEK6.

Similarity:
Belongs to the bZIP family.
Contains 1 bZIP (basic-leucine zipper) domain.

SWISS:
P18848

Gene ID:
468

Database links:

Entrez Gene: 468 Human

Entrez Gene: 11911 Mouse

Entrez Gene: 79255 Rat

NCBI: 33469976 Human

Omim: 604064 Human

SwissProt: P18848 Human

SwissProt: Q96AQ3 Human

SwissProt: Q06507 Mouse

SwissProt: Q5U4B2 Mouse

SwissProt: Q8CF69 Mouse

SwissProt: Q9ES19 Rat

Unigene: 496487 Human

Unigene: 641 Mouse

Unigene: 2423 Rat



Picture

Sample:
Embryo(Mouse) lysate at 30ug;
Eye(Mouse) lysate at 30 ug;
Primary: Anti-ATF4 (SL1531R) at 1:300 dilution;
Secondary: HRP conjugated Goat-Anti-Rabbit IgG(bse-0295G) at 1: 5000 dilution;
Predicted band size : 38kD
Observed band size : 38kD
Sample:
Lane 1: Mouse Spleen Lysates
Lane 2: Mouse Lung Lysates
Lane 3: Mouse Brain Lysates
Lane 4: Rat Brain Lysates
Lane 5: Human Hela cell Lysates
Lane 6: Human HepG2 cell Lysates
Primary: Anti-ATF4 (SL1531R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 38kDa
Observed band size: 50kDa
Sample:
Cerebrum (Rat) Lysate at 40 ug
Cerebrum (Mouse) Lysate at 40 ug
Primary: Anti-ATF4 (SL1531R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 37’50 kD
Observed band size: 50 kD
Paraformaldehyde-fixed, paraffin embedded (human breast carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat spleen); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat lung); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Human breast cancer); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (human brain glioma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (Mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (ATF4) Polyclonal Antibody, Unconjugated (SL1531R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructions and DAB staining.
Tissue/cell: human rectal carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min;
Incubation: Anti-ATF4 Polyclonal Antibody, Unconjugated(SL1531R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Tissue/cell: human gastric carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min;
Incubation: Anti-ATF4/CREB-2 Polyclonal Antibody, Unconjugated(SL1531R) 1:200, overnight at 4℃, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
U2OS cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (ATF4) polyclonal Antibody, Unconjugated (SL1531R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (ATF4) polyclonal Antibody, Unconjugated (SL1531R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Blank control (blue line): Hela
Primary Antibody (green line): Rabbit Anti-Bid antibody (SL1531R)
Dilution: 0.2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC
Dilution: 1μg /test.
Protocol
The cells were fixed with 70% ethanol (Overnight at 4℃) and then permeabilized with 90% ice-cold methanol for 30 min on ice. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
Blank control:Mouse spleen.
Primary Antibody (green line): Rabbit Anti-ATF4 Alpha antibody (SL1531R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.
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