Overview

  • Product name
    Anti-14-3-3 epsilon antibody - Aminoterminal end
    See all 14-3-3 epsilon primary antibodies
  • Description
    Rabbit polyclonal to 14-3-3 epsilon - Aminoterminal end
  • Host species
    Rabbit
  • Specificity
    Raised against an N-terminal epitope which is N-acetylated as in the normal protein therefore may not react with recombinant protein expressed in E. coli that is not N-acetylated.
  • Tested applications
    Suitable for: ICC/IF, IHC-P, WB, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Chicken, Cow, Human
    Predicted to work with: Mammals
  • Immunogen

    Synthetic peptide from within N-terminus

Properties

Applications

Our Abpromise guarantee covers the use of ab43057 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 1/400.
IHC-P Use at an assay dependent concentration.
WB 1/3000. Detects a band of approximately 29 kDa (predicted molecular weight: 29 kDa).
ELISA Use at an assay dependent concentration.

Target

  • Function
    Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathway. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner.
  • Sequence similarities
    Belongs to the 14-3-3 family.
  • Cellular localization
    Cytoplasm. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt
  • Database links
  • Alternative names
    • 14 3 3 E antibody
    • 14 3 3 epsilon antibody
    • 14 3 3E antibody
    • 14-3-3 protein epsilon antibody
    • 14-3-3E antibody
    • 1433E_HUMAN antibody
    • Epididymis luminal protein 2 antibody
    • FLJ45465 antibody
    • FLJ53559 antibody
    • HEL2 antibody
    • KCIP 1 antibody
    • KCIP1 antibody
    • MDCR antibody
    • MDS antibody
    • Mitochondrial import stimulation factor L subunit antibody
    • Protein kinase C inhibitor protein1 antibody
    • Tyrosine 3 monooxygenase/tryptophan 5 monooxygenase activation protein, epsilon antibody
    • Tyrosine 3 monooxygenase/tryptophan 5 monooxygenase activation protein, epsilon polypeptide antibody
    • Tyrosine 3/tryptophan 5 monooxygenase activation protein epsilon polypeptide antibody
    • YWHAE antibody
    see all

Images

  • ICC/IF image of ab43057 stained PC12 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 (ab43057, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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.

  • Ab43057 staining human normal placenta tissue. Staining is localised to cytoplasm.
    Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
    Sections were stained using an automated system (DAKO Autostainer Plus ), at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT Link. . Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
  • All lanes : Anti-14-3-3 epsilon antibody - Aminoterminal end (ab43057) at 1/500 dilution

    Lane 1 : Human brain tissue lysate - total protein (ab29466)
    Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 29 kDa
    Observed band size: 32 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 19 kDa, 74 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 30 seconds

References

This product has been referenced in:
See all 2 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Answer

Thanks so much for the additional details.
It seems that your western blotting experiments are working as I would expect. One typically can not see differences of just 1kDa using western blotting. Western blotting does not have that degree of resolution. So in your transfected samples you are most likely detecting both endogenous and exogenous protein but the signals overlap.
I hope this is helpful. Please contact me again if you have any further questions.

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Question

DESCRIPTION OF THE PROBLEM I transfect RAW cells with a FLAG-tagged 14-3-3epsilon and then check the overexpression with Western blot with 1. this N-terminal 14-3-3epsilon antibody, 2. flag antibody. My negative controls are 1. nontransfected cells, 2. empty vector transfected cells. I detec 14-3-3epsilon at about 29KDa (right in the middle between 25KDa & 35KDa bands) however when I reblot with FLAG antibody, I see that the detected band is slightly closer to 35KDA (which is expected, 1KDa increase should be due to FLAG tag). My question is though, why can I not see a second band with this antibody on my western blots? Is it possible for the FLAG tag to cover the N-terminal region so that I cannot detect the recombinant protein?
SAMPLE I use RAW 264.7 cells, and my samples are whole cell lysates as well as cytoplasmic and nuclear extracts. The recombinant protein is FLAG-14-3-3 Epsilon, Jetprime transfection kit
PRIMARY ANTIBODY ab43057. 1:1000 dilution, 1hr incubation at room temp,3x5min wash
DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED no positive control. 2 negative controls: empty vector transfection and nontransfection
ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION I use RIPA buffer for whole cell lysates. For Cytoplasmic extracts:10 mM HEPES, pH 7.9, with 1.5 mM MgCl2 and 10 mM KCl For resuspension of nuclear pellet: 20 mM HEPES, pH 7.9, with 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, and 25% (v/v) Glycerol We use Sigma protease inhibitor cocktail and PMSF
AMOUNT OF PROTEIN LOADED 15ug/lane
ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE 10%
TRANSFER AND BLOCKING CONDITIONS Transfer 2 hrs, blocking with milk for 1 hr SECONDARY ANTIBODY 1:1000 dilution, 1hr room temp. incubation, 3x5min wash
HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes
DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

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Answer

Thank you for submitting your completed questionnaire.
Please correct me if I am incorrect but you just have the one construct, the flag tagged 14-3-3epsilon protein.
Do you see a band between the 25KDa & 35KDa band in your negative control samples?
Thanks in advance for the additional information.

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Question
Answer

Thank you for your patience.

For ab43057, we might potentially have an immunogen peptide available, but I do not yet have any further details at the moment. I just wanted to let you know, and will be in touch with more details as soon as I have them.

Thank you for your understanding and patience.

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Question
Answer

Thank you for contacting us.

I have heard back for ab63635, ab69592:

Unfortunately, for these 2 antibodies the immunogen is not available for sale.

As for ab97273 and ab43057, I am still waiting for a reply and will let you know as soon as I hear back from the lab.

Thank you for your patience and understanding.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (MCF7 Breast Cancer cell line)
Loading amount
20 µg
Specification
MCF7 Breast Cancer cell line
Gel Running Conditions
Reduced Denaturing (12 %)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Jun 30 2011

Question
Answer

Thank you for your patience.

I have received more information from that lab regarding the availability of the immunogen peptide for ab43057.

Unfortunately, due tolicensing issues, we are not able to add the immunogen peptide to our catalog. I'm very sorry about this and that I am not able to help you further in this regard.

Please do not hesitate to contact us if you need any more advice or information.

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