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Key Product Details

Validated by

Biological Validation

Species Reactivity

Human, Mouse, Rat, Primate

Applications

Flow (Intracellular), Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free

Concentration

1 mg/ml

Product Summary for GADD153/CHOP Antibody - BSA Free

Immunogen

A synthetic peptide made to an internal portion of the human CHOP/GADD153 protein (between residues 100-150) [UniProt P35638]

Reactivity Notes

Mouse reactivity reported in scientific literature (PMID:32814096).

Localization

Cytoplasm when unactivated, translocation to nucleus upon activation.

Marker

ER Stress Marker

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Scientific Data Images for GADD153/CHOP Antibody - BSA Free

Western Blot: GADD153/CHOP Antibody - BSA Free [NBP2-13172] - GADD153/CHOP Antibody [NBP2-13172] - NASTRp induces ER stress and eventually leads to cell death with Bim up-regulation. NASTRp induced ER stress and activated UPR led to apoptosis with Bim induction in NSCLC cell lines. Cells were treated with the indicated concentrations of NASTRp for 24 hours and cell lysates were subjected to SDS-PAGE/Western blot analysis using the indicated antibodies. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0122628) licensed under a CC-BY license.
Immunocytochemistry/Immunofluorescence: GADD153/CHOP Antibody [NBP2-13172] - HeLa cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.5% Triton X-100 in PBS for 5 minutes. The cells were incubated with anti-GADD153/CHOP Antibody NBP2-13172 at 1 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.
Western Blot: GADD153/CHOP Antibody - BSA Free [NBP2-13172] - GADD153/CHOP Antibody [NBP2-13172] - Effects of psoralen on ER-stress related protein expression in SMMC7721. Western blot assays showing the effects of psoralen and thapsigargin on ER-stress related protein expression. Image collected and cropped by CiteAb from the following publication (https://biolres.biomedcentral.com/articles/10.1186/s40659-019-0241-8), licensed under a CC-BY license.

Applications for GADD153/CHOP Antibody - BSA Free

Application
Recommended Usage

Flow (Intracellular)

2-5 ug/million cells

Flow Cytometry

2-5 ug/million cells

Immunocytochemistry/ Immunofluorescence

1-5 ug/ml

Immunohistochemistry

1:400

Immunohistochemistry-Paraffin

1:400

Western Blot

1:1000
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 1 review rated 5 using NBP2-13172 in the following applications:

Published Applications

Read 23 publications using NBP2-13172 in the following applications:

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: GADD153

GADD153 also known as DNA damage-inducible transcript 3 protein (DDIT3), C/EBP-homologous protein (CHOP), CHOP10, and CEBPZ, is a growth arrest and DNA damage inducible gene that encodes a C/EBP-related protein. GADD153 is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors. GADD153 can be found in the nucleus and its expression is induced by cellular stresses including nutrient deprivation, endoplasmic reticulum stress, and metabolic perturbations. The GADD153 protein functions as a dominant-negative inhibitor that inhibits DNA-binding activity with other C/EBP members, such as C/EBP and LAP (liver activator protein), by forming heterodimers. Fusion of GADD153 and FUS on chromosome 16 or EWSR1 on chromosome 22, induced by translocation, generates chimeric proteins in myxoid liposarcomas, also known as Ewing sarcoma. GADD153 is implicated in adipogenesis and promotes apoptosis in cells.

Long Name

Growth Arrest and DNA Damage-inducible Protein GADD153

Alternate Names

CEBP zeta, CHOP, CHOP10, DDIT3

Entrez Gene IDs

1649 (Human); 13198 (Mouse); 29467 (Rat)

Gene Symbol

DDIT3

UniProt

Product Documents for GADD153/CHOP Antibody - BSA Free

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for GADD153/CHOP Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Protocols

View specific protocols for GADD153/CHOP Antibody - BSA Free (NBP2-13172):

Protocol for Flow Cytometry Intracellular Staining
Sample Preparation.
1. Grow cells to 60-85% confluency. Flow cytometry requires between 2 x 105 and 1 x 106 cells for optimal performance.
2. If cells are adherent, harvest gently by washing once with staining buffer and then scraping. Avoid using trypsin as this can disrupt certain epitopes of interest. If enzymatic harvest is required, use Accutase, Collagenase, or TrypLE Express for a less damaging option.
3. Reserve 100 uL for counting, then transfer cell volume into a 50 mL conical tube and centrifuge for 8 minutes at 400 RCF.
a. Count cells using a hemocytometer and a 1:1 trypan blue exclusion stain to determine cell viability before starting the flow protocol. If cells appear blue, do not proceed.
4. Re-suspend cells to a concentration of 1 x 106 cells/mL in staining buffer (NBP2-26247).
5. Aliquot out 100 uL samples in accordance with your experimental samples.

Tip: When cell surface and intracellular staining are required in the same sample, it is advisable that the cell surface staining be performed first since the fixation and permeabilization steps might reduce the availability of surface antigens.

Intracellular Staining.
Tip: When performing intracellular staining, it is important to use appropriate fixation and permeabilization reagents based upon the target and its subcellular location. Generally, our Intracellular Flow Assay Kit (NBP2-29450) is a good place to start as it contains an optimized combination of reagents for intracellular staining as well as an inhibitor of intracellular protein transport (necessary if staining secreted proteins). Certain targets may require more gentle or transient permeabilization protocols such as the commonly employed methanol or saponin-based methods.
Protocol for Cytoplasmic Targets:
1. Fix the cells by adding 100 uL fixation solution (such as 4% PFA) to each sample for 10-15 minutes.
2. Permeabilize cells by adding 100 uL of a permeabilization buffer to every 1 x 106 cells present in the sample. Mix well and incubate at room temperature for 15 minutes.
a. For cytoplasmic targets, use a gentle permeabilization solution such as 1X PBS + 0.5% Saponin or 1X PBS + 0.5% Tween-20.
b. To maintain the permeabilized state throughout your experiment, use staining buffer + 0.1% of the permeabilization reagent (i.e. 0.1% Tween-20 or 0.1% Saponin).
3. Following the 15 minute incubation, add 2 mL of the staining buffer + 0.1% permeabilizer to each sample.
4. Centrifuge for 1 minute at 400 RCF.
5. Discard supernatant and re-suspend in 100 uL of staining buffer + 0.1% permeabilizer.
6. Add appropriate amount of each antibody (eg. 1 test or 1 ug per sample, as experimentally determined).
7. Mix well and incubate at room temperature for 30 minutes- 1 hour. Gently mix samples every 10-15 minutes.
8. Following the primary/conjugate incubation, add 1-2 mL/sample of staining buffer +0.1% permeabilizer and centrifuge for 1 minute at 400 RCF.
9. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
10. Add appropriate amount of secondary antibody (as experimentally determined) to each sample.
11. Incubate at room temperature in dark for 20 minutes.
12. Add 1-2 mL of staining buffer and centrifuge at 400 RCF for 1 minute and discard supernatant.
13. Wash twice by re-suspending cells in staining buffer (2 mL for tubes or 200 uL for wells) and centrifuging at 400 RCF for 5 minutes. Discard supernatant.
14. Resuspend in an appropriate volume of staining buffer (usually 500 uL per sample) and proceed with analysis on your flow cytometer.

Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.
[[URL:https://www.novusbio.com/products/gadd153-chop-antibody_nbp2-13172]][[C… antibody]]
Immunohistochemistry-Paraffin Embedded Sections Protocol

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes.

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in wash buffer for 5 minutes.
3. Block each section with 100-400 ul blocking solution for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated diluted secondary antibody. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
8. Add 100-400 ul Streptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in deionized water.
12. Counterstain sections in hematoxylin.
13. Wash sections in deionized water two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.
[[URL:https://www.novusbio.com/products/gadd153-chop-antibody_nbp2-13172]][[C… antibody]]
Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 40 ug of total protein per lane.
2. Transfer proteins to membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain according to standard Ponceau S procedure (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot.
5. Block the membrane using standard blocking buffer for at least 1 hour.
6. Wash the membrane in wash buffer three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate 1 hour at room temperature.
8. Wash the membrane in wash buffer three times for 10 minutes each.
9. Apply the diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions.
Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.
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