Anti-ATF-4 antibody (ab23760)

Submit a review Q&A (5)References (18)

Overview

  • Product name
    Anti-ATF-4 antibody
    See all ATF-4 primary antibodies
  • Description
    Rabbit polyclonal to ATF-4
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human ATF-4 aa 300 to the C-terminus (C terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).
    Database link: P18848
    (Peptide available as ab24034)

  • Positive control
    • ATF-4 tagged recombinant protein This antibody gave a positive signal in the following Methanol fixed cell lines: MCF-7.

Properties

Applications

Our Abpromise guarantee covers the use of ab23760 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
ICC/IF Use a concentration of 5 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 38 kDa (predicted molecular weight: 39 kDa).

Target

  • Function
    Transcriptional activator. Binds the cAMP response element (CRE) (consensus: 5'-GTGACGT[AC][AG]-3'), a sequence present in many viral and cellular promoters. It binds to a Tax-responsive enhancer element in the long terminal repeat of HTLV-I.
  • Sequence similarities
    Belongs to the bZIP family.
    Contains 1 bZIP domain.
  • Cellular localization
    Cytoplasm. Cell membrane. Nucleus. Colocalizes with GABBR1 in hippocampal neuron dendritic membranes.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • Activating transcription factor 4 antibody
    • ATF 4 antibody
    • ATF4 antibody
    • ATF4 protein antibody
    • ATF4_HUMAN antibody
    • cAMP-dependent transcription factor ATF-4 antibody
    • cAMP-responsive element-binding protein 2 antibody
    • CREB 2 antibody
    • CREB-2 antibody
    • CREB2 antibody
    • Cyclic AMP dependent transcription factor ATF 4 antibody
    • Cyclic AMP response element binding protein 2 antibody
    • Cyclic AMP-dependent transcription factor ATF-4 antibody
    • Cyclic AMP-responsive element-binding protein 2 antibody
    • DNA binding protein TAXREB67 antibody
    • DNA-binding protein TAXREB67 antibody
    • Tax Responsive Enhancer Element B67 antibody
    • Tax-responsive enhancer element-binding protein 67 antibody
    • TaxREB67 antibody
    • TXREB antibody
    see all

Images

  • Western blot - Anti-ATF-4 antibody (ab23760)
    Western blot - Anti-ATF-4 antibody (ab23760)
    Anti-ATF-4 antibody (ab23760) at 1 µg/ml + ATF4 tagged recombinant protein at 0.1 µg

    Secondary
    IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size: 39 kDa
    Observed band size: 76 kDa
    why is the actual band size different from the predicted?



    Ab23760 gave a positive signal against the tagged recombinant ATF4 protein which is known to run at 76 kDa.
  • Immunocytochemistry/ Immunofluorescence - Anti-ATF-4 antibody (ab23760)
    Immunocytochemistry/ Immunofluorescence - Anti-ATF-4 antibody (ab23760)

    ICC/IF image of ab23760 stained MCF-7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab23760 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • Zhu X  et al. lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression. Int J Mol Med 43:345-357 (2019). Read more (PubMed: 30431065) »
  • Yang F  et al. ER-stress regulates macrophage polarization through pancreatic EIF-2alpha kinase. Cell Immunol N/A:N/A (2018). Read more (PubMed: 30594305) »
See all 18 Publications for this product

Publishing research using ab23760? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-5 of 5 Abreviews or Q&A

Question

Please could you let us know when we should receive the antibodies in substitution?   Thank you very much

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Answer

I have received confirmation that these 3 replacement vials will be included in the next shipment to Aurogene. I hope this is helpful. Please contact me again if you have any further questions.

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Question

our customer told us that a control can not solve her problems. It could be useful only for the difference between the expected and observed molecular weight (in our case relative to RANKL antibody), but also in that case she's sure that there was no problem in gel running and the molecular weights were indeed those identified by the standard, because after revealing RANKL they have also revealed actin, with the band showing the expected length. Moreover, your the use of a control does not resolve the problem of OPG and ATF4 aspecificity. For this reason, she has requested the substitution of items ab10962, ab23760, ab45039. Please could you let us know when you should ship us these antibodies in substitution? Our customer needs them urgently.  

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Answer

Thanks for your reply. It seems that, as part of their own internal QC, the customer has already peformed the control I suggested. This is great as it does appear clear now that these 3 antibodies are not working as expected. I would be happy to request the replacement antibodies the customer asked for: 1 vial each of ab10962, ab23760, ab45039. So I can process the request, please let me know the purchase order number or Abcam order reference number associated with the initial order of these 3 antibodies. Thanks!

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Question

What species of bone cells were being tested?   Murine Osteoblasts from calvaria What dilution of primary antibody for each?   OPG 1:100; ATF4 1:400; RANKL 1:500 Was any other antibody used successfully with these same samples?  No How much protein was loaded on the gel? 20 ug   Please, could you tell us when you'll ship us the antibodies in substitution?  

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Answer

Thank you for providing the additional information. The one thing I would like to recommend in this case is to run a "loading control" western blot with the murine osteoblast cell lysate. Examples of targets for this type of control would be proteins that are commonly expressed at high levels in all cell types, such as actin or GAPDH. This would serve as a control in that detection of the expected band size would indicate that all aspects of the lysate preparation and western blot protocol are working as expected. Thanks so much for your troubleshooting efforts! I look forward to hearing how things go.

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Question

the last June our company Aurogene ordered from you the antibodies ab10962 goat polyclonal to Osteoprotegerin, ab23760 rabbit polyclonal to ATF4 and ab45039 mouse monoclonal |12A668| ro RANKL, that didn't work when used by our customer. In attachment we send you the WB images of these antibodies. The experiments were performed using lysates of bone cells that certainly synthesize proteins for which the above antibodies should be specific. As for the first 2, the customer has found a strong aspecificity, while for RANKL he has seen a single band, but the found molecular weight (65 kDa approximately) is much higher than expected (35 kDa). Please, could you substitute as soon as possible these antibodies?

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Answer

 Thank you for your enquiry. Sorry to hear your customer's are having trouble with these antibodies.   What species of bone cells were being tested? What dilution of primary antibody for each? Was any other antibody used successfully with these same samples? How much protein was loaded on the gel? What was the purchase order number or Abcam order reference number associated with these purchases? Thanks in advance for the additional information.

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Question

BATCH NUMBER 158060 ORDER NUMBER 148114 DESCRIPTION OF THE PROBLEM No signal SAMPLE cell extract PRIMARY ANTIBODY Abcam/Rabbit plyclone to ATF-4/1ugm/ml / O/N incubation at 4C DETECTION METHOD ECLPlus POSITIVE AND NEGATIVE CONTROLS USED Positive Control Liver Extract SAMPLE PREPARATION Buffer AMOUNT OF PROTEIN LOADED 50-100 ugm/lane ELECTROPHORESIS/GEL CONDITIONS 10% PAGE gel TRANSFER AND BLOCKING CONDITIONS PBST 5% Non fat Milk SECONDARY ANTIBODY [a competitor]/anti Rabbitdilution is 1:5000 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? I tried 1 hour blocking and O/N with primary ab/ 1hour incubation and 1 hr pr. ab. at 4C and Room temp. and also change the blocking buffer dried milk to BSA with and without Twin-20

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Answer

Thank you for contacting us for technical support and for taking the time to provide your protocol information. I'm sorry to hear you are experiencing problems with this antibody. I have tried to find in the literature references for the presence of ATF4 in liver and have not been successful, you mention it as a positive control and I would be very grateful if you could confirm the source of this information as I currently think that levels in the liver may be low. SwissProt mentions lung and placenta as tissues expressing the protein, and we have used HeLa lysate as our positive control. I would recommend to run this lysate along your samples to make sure this is not the problem. I would also like to recommend to perform a nuclear extraction procedure and to make sure the extraction buffer you currently use is strong enough to break the nucleus, as the protein is there. Could you please also check that the problem is not due to the secondary antibody, and can I check that you have been using reducing conditions? I hope the above recommendations are useful, please do not hesitate to contact me if you still experience problems with the positive control of Hela lysate,

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