Overview

  • Product name
    Anti-DDIT3 antibody [9C8]
    See all DDIT3 primary antibodies
  • Description
    Mouse monoclonal [9C8] to DDIT3
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, IP, ICC, ICC/IF, IHC-Fr, Flow Cyt, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Other Immunogen Type corresponding to DDIT3. A bacterially expressed, mouse DDIT3 fusion protein.

  • Epitope
    ab11419 has been shown to recognize an epitope in the N-terminal region of DDIT3.
  • Positive control
    • ICC/IF: Rat supraoptic nucleus; HeLa cells. WB: HeLa cells treated with tunicamycin (5µg/ml) for 11 hours; Mouse hepatocyte whole cell lysate; Primary human fibroblasts treated with tunicamycin for 10 hours; Mouse 3T3 cells treated with tunicamycin for 24 hours. Flow: HeLa cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab11419 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use a concentration of 1 µg/ml. Detects a band of approximately 31 kDa (predicted molecular weight: 19 kDa). Please note that under normal cellular conditions this protein is not expressed in detectable levels, but is highly upregulated during times of cellular/ER stress. It is strongly recommended to run a positive control along your samples to confirm the expression levels of protein.
IP Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
IHC-Fr 1/100.
Flow Cyt Use 1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

IHC-P Use at an assay dependent concentration. PubMed: 19352619

Target

  • Function
    Inhibits the DNA-binding activity of C/EBP and LAP by forming heterodimers that cannot bind DNA.
  • Involvement in disease
    Note=A chromosomal aberration involving DDIT3 is found in a patient with malignant myxoid liposarcoma. Translocation t(12;16)(q13;p11) with FUS.
  • Sequence similarities
    Belongs to the bZIP family.
    Contains 1 bZIP domain.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • C/EBP homologous protein antibody
    • C/EBP Homology Protein antibody
    • C/EBP zeta antibody
    • C/EBP-homologous protein 10 antibody
    • C/EBP-homologous protein antibody
    • CCAAT/enhancer binding protein homologous protein antibody
    • CEBPZ antibody
    • CHOP 10 antibody
    • CHOP antibody
    • CHOP-10 antibody
    • CHOP10 antibody
    • DDIT 3 antibody
    • DDIT-3 antibody
    • Ddit3 antibody
    • DDIT3_HUMAN antibody
    • DNA Damage Inducible Transcript 3 antibody
    • DNA damage-inducible transcript 3 protein antibody
    • GADD 153 antibody
    • GADD153 antibody
    • Growth Arrest and DNA Damage Inducible Protein 153 antibody
    • Growth arrest and DNA damage inducible protein GADD153 antibody
    • Growth arrest and DNA damage-inducible protein GADD153 antibody
    • MGC4154 antibody
    see all

Images

  • Immunostaining of DDIT3 (green) and AVP (red) in the supraoptic nucleus of 3 days DH and 7 days SL rats shows expression of CHOP in the nuclei of AVP MCNs (magnocellular neurons). DDIT3 was stained using ab11419 at 1/200 dilution.

    The rat  brains were removed and post-fixed overnight in 4% (w/v) PFA followed by 30% (w/v) sucrose prepared in PBS to cryoprotect the tissue prior to freezing over liquid nitrogen. Coronal sections (40 μm) of the forebrain were cut on a cryostat and washed in PBS three times. Sections then were blocked in 5% fetal bovine serum prepared in 0.25% (v/v) TritonX/PBS (PBST) for 30 min and then incubated with appropriate primary antibodies at 4°C for 48 hours.

    DH = Complete fluid deprivation.
    SL = Salt loading by drimking 2% salt solution

  • Western blot analysis of HeLa cell lysates using DDIT3 monoclonal antibody (ab11419). HeLa cells were left untreated (-) or treated with tunicamycin (5μg/ml) for 11 hours (+). Membranes containing whole cell extracts were blocked with 5% milk in TBST (blocking buffer) and probed overnight at 4oC with ab11419 (diluted 1:2000 in blocking buffer) followed by a goat anti-mouse IgG-HRP secondary antibody (diluted in blocking buffer) and a chemiluminescent substrate. Membranes were also probed for actin as a loading control.

  • ab11419 staining GADD153 in human HeLa cells by Immunocytochemistry/ Immunofluorescence. The cells were fixed in paraformaldehyde, permeabilised in 0.5% Triton X and then blocked using 3% BSA for 1 hour at 24°C. Samples were then incubated with primary antibody at a 1/100 dilution for 1 hour at 24°C. The secondary antibody used was a goat anti-mouse IgG conjugated to Alexa Fluor® 488 (green) used at a 1/1000 dilution.
    HeLa cells treated with 20uM Tunicamycin for 15 minutes before fixation.

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab11419 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab11419, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b  (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • All lanes : Anti-DDIT3 antibody [9C8] (ab11419) at 1/1000 dilution

    All lanes : Mouse hepatocyte whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : HRP-conjugated goat anti-mouse IgG polyclonal at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 19 kDa
    Observed band size: 27 kDa
    why is the actual band size different from the predicted?


    Exposure time: 5 minutes


    Treated with 20µg/ml poly(I:C).

    See Abreview

  • Western blot of endogenous DDIT3 from primary Human fibroblasts using ab11419.
    Lane 1: Untreated cells
    Lane 2: Cells treated with tunicamycin for 10 hours

  • All lanes : Anti-DDIT3 antibody [9C8] (ab11419) at 1/500 dilution (in TBST + 2.5% milk for 16 hours at 4°C)

    Lane 1 : Whole cell lystate of Mouse 3T3 cells
    Lane 2 : Whole cell lystate of Mouse 3T3 cells treated with tunicamycin for 24 hours

    Lysates/proteins at 50 µg per lane.

    Secondary
    All lanes : An HRP-conjugated Goat anti-mouse IgG monoclonal at 1/2000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 19 kDa
    Observed band size: 31 kDa why is the actual band size different from the predicted?


    Exposure time: 2 minutes


    Blocking Step: 5% Milk for 2 hours at 22°C

    See Abreview

  • ab11419 staining DDIT3 in SKNSH cells treated with deltamethrin (ab141019), by ICC/IF. Increase of DDIT3 expression correlates with increased concentration of deltamethrin, as described in literature.
    The cells were incubated at 37°C for 48 hours in media containing different concentrations of ab141019 (deltamethrin) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab11419 (10 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

References

This product has been referenced in:
  • Leipnitz G  et al. Evaluation of mitochondrial bioenergetics, dynamics, endoplasmic reticulum-mitochondria crosstalk, and reactive oxygen species in fibroblasts from patients with complex I deficiency. Sci Rep 8:1165 (2018). WB ; Human . Read more (PubMed: 29348607) »
  • Xie Q  et al. TAT-fused IP3R-derived peptide enhances cisplatin sensitivity of ovarian cancer cells by increasing ER Ca2+ release. Int J Mol Med 41:809-817 (2018). Read more (PubMed: 29207009) »
See all 72 Publications for this product

Customer reviews and Q&As

1-10 of 34 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (brain tissue)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate ph 6
Permeabilization
No
Specification
brain tissue
Blocking step
Normal Goat Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 26°C
Fixative
Formaldehyde

Herr Dr. Markus Kipp

Verified customer

Submitted Aug 17 2018

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Sheep Tissue sections (Intestinal slides, Ileum)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer pH 6.0
Permeabilization
No
Specification
Intestinal slides, Ileum
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 12 2017

Application
ChIP
Sample
Human Cell lysate - whole cell (Heptocyte)
Negative control
Untreated cells
Specification
Heptocyte
Detection step
Semiquantitative PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Positive control
Efavirenz

Abcam user community

Verified customer

Submitted Oct 07 2016

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (HEPATOCYTES)
Specification
HEPATOCYTES
Treatment
20 ug/ml poly(I:C)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Jul 09 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (SH-SY5Y cell)
Loading amount
100 µg
Specification
SH-SY5Y cell
Treatment
25um cadmium
Gel Running Conditions
Non-reduced Denaturing
Blocking step
Milk as blocking agent for 4 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 08 2013

Answer

Thank you for your reply.
Your credit note ID is ***.
I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.
Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.
The credit note ID is for your reference only and does not automatically guarantee the credit.
I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

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Question

1.Antibody storage conditions (temperature/reconstitution etc) -20°C 2. Please describe the problem (high background, no staining etc). no staining 3. On what material are you testing the antibody in IHC Species: human Cell Iine or tissue:placenta 4. How did you fix the samples? Ethanol, methanol: (was it ice cold) Acetone: (was it ice cold) Paraformaldehyde (percentage, perfusion fixed on not):10% formaldehyde solution,no perfusion Fixation time:12-24hours Fixation temperature: room temperature 5. For formalin-fixed paraffin embedded tissue: did you apply antigen retrieval step? Enzymatic method Heat mediated technique 6. Blocking steps: For HRP detection method: did you block endogenous peroxidases: 3%H2O2 10minutes How did you block the unspecific binding sites: serum, BSA, Milk, other: BSA For how long:10-20minutes 7. Primary antibody Specification (in which species was it raised against): Mouse At what dilution(s) have you tested this antibody:1:100,1:150,1:200,1:300 What dilution buffer was used:PBS Incubation time:2 huors room tempture or 4°C one night Incubation temperature: What washing steps were done: PBS 3 times 8. Secondary antibody - Specification (in which species was it raised against): goat anti-mouse - At what dilution(s) have you tested this antibody: - What dilution buffer was used: no - Incubation time:20 minutes - Incubation temperature: room tempture - What washing steps were done: PBS 3 times - Do you know whether the problems you are experiencing come from the secondary? No 9. What detection method are you using? DAB 10. Did you apply positive and negative controls along with the samples? Please specify. Yes! Placental tissue stained breast cancer sections were stained as positive control, with PBS instead of antibody as a negative control 11. Optimization attempts - How many times have you tried the IHC? 10 - Do you obtain the same results every time? YES - What steps have you altered? we have tried the dilution of Primary antibody from 1:100 to 1:300.and changed incubation conditions(2huors room tempture or 4°C one night) ,and proglonged staining(from 2 minutes to 7 minutes)

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Answer

Thank you for contacting Abcam.
I am sorry about the issues that you have been having with ab11419 in IHC-P on human samples. The antibody is covered under our Abpromise for six months and is guaranteed to work in IHC-P on human samples. Based on the information that you have provided, I do not think that there is any protocol variations that I could provide that would resolve the situation. Therefore I would like to offer to either replace or refund the cost of the antibody.
An alternative that should work for you is, ab59396:
https://www.abcam.com/GADD153-antibody-ab59396.html
Please let me know how you would you like to continue and if you could also provide either the Abcam order number or the PO# used to purchase the antibody that would be very helpful.
I look forward to your reply.

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Answer

Thank you for providing that information.

As I mentioned previously, with the exception of ab79483, we do guarantee that these antibodies should work in western blot on rat tissue. If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

For ab37151, although the predicted molecular weight is 29kDa, we have some images on our webages (and attached) that show that in some samples the antibody recognizes a band of about 42kDa, which is about the size of the band that you are seeing. The difference in molecular weights could be due to pos-translational modifications, such as glycosylation etc. Therefore, I believe that this antibody is actually working well for you.

For ab50354, there is potentially a band (below the 35kDa marker) that could be the correct product, but due to the high background, it is unclear. I would advice, blocking in BSA, and then incubating the primary antibody (also in BSA) at 1/500, with overnight incubation at 4C. Hopefully that will improve the quality of the results. If not, then under the terms of our Abpromise, I will be happy to replace or refund the antibody for you.

For ab79483, as we had not tested this antibody in rat tissue, we could not guarantee that it would work for that species. I will be happy to help troubleshoot this, but it may be that the antibody is unsuitable for working in this species. You could try not only increasing the amount of total protein you are loading into the wells, but you could also increase the primary antibody concentration to 1/500.

For ab11419, again the predicted and actual molecular weights are different, but the band you are seeing just above 35kDa, may be the correct signal. A little higher than others have seen, but potentially the correct band. To confirm this, I would advise, rerunning the el, but this time let it run a little longer to get some better separation of these bands, and then as I suggested for ab50354, try blocking in BSA to help reduce the background.

Please let me know if the protocol variations do not prove to be successful, or if there is anything else that I can help you with.

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Answer

Thank you for contacting Abcam.

I am sorry that you are having some issues with our antibodies. All of the antibodies you mentioned should work in western blot in various species, however only ab30315 is known to work in IHC-P. Below is a list of what applications and species we guarantee each antibody to work in:

ab37151 - ICC, WB - Mouse, Rat, Human
ab30315 - IP, WB, ICC/IF, IHC-P - Mouse, Rat, Human, Xenopus laevis
ab50354 - WB, ICC/IF - Mouse, Rat, Human
ab81959 - WB - Mouse, Rat, Human
ab79483 - WB, ELISA, IHC-P - Human
ab11419 - WB, IP, ICC/IF, IHC - Mouse, Rat, Human

To be able to help you further, would you be able to label the western blot images you sent with the molecular weights and also with what is in each lane. This way I will be able to understand the images that you sent.

Also, would you be able to answer the questions below, regarding the protocol that you are using:

1 - What sample type are you using and from what species?

2 - How much sample are you loading?

3 - What type of blocking agent are you using?

4 - What primary antibody concentrations have you tried and how long do you incubate for?

We do guarantee our antibodies to work as stated on the datasheets and if we cannot get the antibodies to work for you as we state on the datasheet, then I would be happy to replace or refund the cost of the antibody.

I look forward to your reply.

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Answer

Thank you for your reply.


I am inclined to believe that the issue is with the samples themselves. It is very unusual for 3 different antibodies to detect bands mainly above 100kDa when the expected MW should be around 25kDa. How long are the samples boiled before loading? In reading your protocol, I could only find 1 minute, which is not sufficient. Boiling 5-10 min is advised to completely denature the proteins.


How do the samples look if you stain the gel with Coomassie? or Ponceau S?

Furthermore, for at least GADD153, expression must be induced in your samples. Did you do this? Also, ab54740 is only validated for human and may not work with your MEFs. Are you running any sort of a loading control to confirm that the procedure is working with other antibodies?

I hope this information is helpful. Please do not hesitate to contact me if you have additional questions.

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1-10 of 34 Abreviews or Q&A

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