Anti-alpha Adaptin antibody [AP6] (ab2730)

Reviews (10)Q&A (4)References (18)

Overview

  • Product name

    Anti-alpha Adaptin antibody [AP6]
    See all alpha Adaptin primary antibodies

  • Description

    Mouse monoclonal [AP6] to alpha Adaptin

  • Host species

    Mouse

  • Specificity

    Detects assembly polypeptide 2 (AP2) It recognizes the products of both alpha-adaptin genes, alpha A and alpha C as well as an alternatively spliced isoform of alpha A found in neurons.

  • Tested applications

    Suitable for: ICC/IF, ICC, Inhibition Assay, Electron Microscopy, ELISA, Blocking, IP, WB, IHC-FoFr, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Hamster, Cow, Human, Drosophila melanogaster
    Predicted to work with: Non human primates

  • Immunogen

    Other Immunogen Type corresponding to alpha Adaptin. Purified adaptors.

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.

  • Storage buffer

    Preservative: 0.05% Sodium azide
    Constituent: PBS
  • Concentration information loading...

  • Purity

    Affinity purified

  • Clonality

    Monoclonal

  • Clone number

    AP6

  • Isotype

    IgG1

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab2730 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.
ICC Use at an assay dependent concentration.
Inhibition Assay Use at an assay dependent concentration.
Electron Microscopy Use at an assay dependent concentration. PubMed: 23372794
ELISA Use at an assay dependent concentration.
Blocking Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB 1/1000. This antibody detects an ~100 kDa doublet representing the two isoforms of alpha-adaptin in rat brain, though alpha C is the dominant form seen. Microinjection of cells with this antibody results in variable blockade of endocytosis.
IHC-FoFr Use at an assay dependent concentration.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

Target

  • Relevance

    Clathrin mediated endocytosis is the pathway by which many receptors for nutrients and hormones are internalized to be recycled or down regulated. During formation of clathrin coated membranes, clathrin co assembles with heterotetrameric molecules known as assembly polypeptides (APs) or adaptors which form a layer of protein coat between the clathrin lattice and the membrane. There are two characterized adaptors AP1 and AP2. AP1 is associated with clathrin coated vesicles at the trans Golgi network and AP2 is associated with the endocytic clathrin coated vesicles at the plasma membrane and has been shown to specifically interact with Shc and EGF receptor. AP2 is composed of four subunits, two separate 100 kDa gene products with similar domain structures (alpha and beta adaptin) and a 50 and 17 kDa subunit. There are two alpha adaptin genes, alpha A and alpha C which have a tissue specific pattern of expression.

  • Cellular localization

    Cytoplasmic

  • Database links

    see all

  • Alternative names

    • 100 kDa coated vesicle protein A antibody
    • 100 kDa coated vesicle protein C antibody
    • Adapter related protein complex 2 subunit alpha 1 antibody
    • Adapter related protein complex 2 subunit alpha 2 antibody
    • Adaptin alpha A antibody
    • Adaptin alpha B antibody
    • Adaptin, alpha B antibody
    • Adaptor protein complex AP 2 alpha 1 subunit antibody
    • Adaptor protein complex AP 2 alpha 2 subunit antibody
    • Adaptor protein complex AP 2 subunit alpha 1 antibody
    • Adaptor related protein complex 2 alpha 1 subunit antibody
    • Adaptor related protein complex 2 alpha 2 subunit antibody
    • ADTAA antibody
    • ADTAB antibody
    • Alpha adaptin A antibody
    • Alpha adaptin C antibody
    • Alpha adaptin C; Huntingtin interacting protein J antibody
    • Alpha1 adaptin antibody
    • Alpha2 adaptin antibody
    • AP 2 complex subunit alpha 1 antibody
    • AP 2 complex subunit alpha 2 antibody
    • AP2 alpha antibody
    • AP2A1 antibody
    • AP2A2 antibody
    • CLAPA1 antibody
    • CLAPA2 antibody
    • Clathrin assembly protein complex 2 alpha A large chain antibody
    • Clathrin assembly protein complex 2 alpha C large chain antibody
    • Clathrin associated/assembly/adaptor protein large alpha 1 antibody
    • Clathrin associated/assembly/adaptor protein large alpha 2 antibody
    • HIP 9 antibody
    • HIP9 antibody
    • Huntingtin interacting protein antibody
    • Huntingtin interacting protein HYPJ antibody
    • Huntingtin interacting protein J antibody
    • Huntingtin yeast partner J antibody
    • HYPJ antibody
    • Plasma membrane adaptor HA2/AP2 adaptin alpha A subunit antibody
    • Plasma membrane adaptor HA2/AP2 adaptin alpha C subunit antibody
    see all

Images

  • Immunocytochemistry/ Immunofluorescence - Anti-alpha Adaptin antibody [AP6] (ab2730)
    Immunocytochemistry/ Immunofluorescence - Anti-alpha Adaptin antibody [AP6] (ab2730)
    Immunofluorescent analysis of alpha Adaptin using alpha Adaptin Monoclonal Antibody (AP6) (ab2730) shows staining in U251 Cells. alpha Adaptin (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing alpha Adaptin (ab2730) at a dilution of 1:20 over night at 4C and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.
  • Immunocytochemistry/ Immunofluorescence - Anti-alpha Adaptin antibody [AP6] (ab2730)
    Immunocytochemistry/ Immunofluorescence - Anti-alpha Adaptin antibody [AP6] (ab2730)
    Immunofluorescent analysis of alpha Adaptin using alpha Adaptin Monoclonal Antibody (AP6) (ab2720) shows staining in MCF-7 Cells. alpha Adaptin (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing alpha Adaptin (ab2730) at a dilution of 1:20 over night at 4 C and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.
  • Immunocytochemistry/ Immunofluorescence - Anti-alpha Adaptin antibody [AP6] (ab2730)
    Immunocytochemistry/ Immunofluorescence - Anti-alpha Adaptin antibody [AP6] (ab2730)
    Immunofluorescent analysis of alpha Adaptin using alpha Adaptin Monoclonal Antibody (AP6) (ab2730) shows staining in Hela Cells. alpha Adaptin (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing alpha Adaptin (ab2720) at a dilution of 1:20 over night at 4 C and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.
  • Immunocytochemistry/ Immunofluorescence - Anti-alpha Adaptin antibody [AP6] (ab2730)
    Immunocytochemistry/ Immunofluorescence - Anti-alpha Adaptin antibody [AP6] (ab2730)
    ICC/IF image of ab2730 stained MCF7 cells. The cells were 4% formaldehyde fixed (10 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 (ab2730, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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.

  • Flow Cytometry - Anti-alpha Adaptin antibody [AP6] (ab2730)
    Flow Cytometry - Anti-alpha Adaptin antibody [AP6] (ab2730)

    Overlay histogram showing MCF7 cells stained with ab2730 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab2730, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Western blot - Anti-alpha Adaptin antibody [AP6] (ab2730)
    Western blot - Anti-alpha Adaptin antibody [AP6] (ab2730)Image from Li Y et al, J Biol Chem. 2012 Jan 2;287(1):523-30. Epub 2011 Nov 11, Fig 2. DOI 10.1074/jbc.M111.306183 January 2, 2012 The Journal of Biological Chemistry, 287, 523-530.
    ab2730 used in Western Blot at a 1/1000 dilution.
    Sucrose density gradient fractionation of detergent resistant cell lysates from WT and DHHC5-GT murine neuronal stem cells. The isolation of detergent-resistant membranes was performed according to a previously published protocol. NSCs growing in growth medium (~300 µl of packed cells) were harvested and washed three times with ice-cold PBS. The cells were gently resuspended by pipetting up and down in 700 µl of 1% Triton X-100 in HEPES buffer (25 mm HEPES-HCl, pH 6.5, 150 mm NaCl, 1 mm EDTA, and protease inhibitor mixture), homogenized using a Teflon-coated Dounce homogenizer (30 strokes), then incubated on ice for 30 min, and fractionated on a sucrose density gradient. The fractions were collected from top (fraction 1) to bottom (fraction 13) and analyzed by immunoblotting. The results shown were from one of two independent experiments giving similar results. a-Adaptin is a marker for clathrin-coated vesicles.

References

This product has been referenced in:

See all 18 Publications for this product

Publishing research using ab2730? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 14 Abreviews or Q&A

Immunocytochemistry/ Immunofluorescence abreview for Anti-alpha Adaptin antibody [AP6]

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (undifferentiated Human ES cells on MEFs)
Permeabilization
Yes - in blocking buffer
Specification
undifferentiated Human ES cells on MEFs
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde
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Abcam user community

Verified customer

Submitted Feb 01 2016

Western blot abreview for Anti-alpha Adaptin antibody [AP6]

 Below Average
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (Undifferentiated Human ES cells on MEFs)
Gel Running Conditions
Non-reduced Denaturing (12.5%)
Loading amount
20 µg
Specification
Undifferentiated Human ES cells on MEFs
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C
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Abcam user community

Verified customer

Submitted Jan 25 2016

Immunocytochemistry/ Immunofluorescence abreview for Anti-alpha Adaptin antibody [AP6]

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEPG2)
Specification
HEPG2
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: rt°C
Fixative
Paraformaldehyde
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Abcam user community

Verified customer

Submitted Nov 18 2015

Western blot abreview for Anti-alpha Adaptin antibody [AP6]

 Below Average
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (HEPG2)
Gel Running Conditions
Reduced Denaturing (12.5)
Loading amount
20 µg
Specification
HEPG2
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 16°C
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Abcam user community

Verified customer

Submitted Nov 13 2015

Immunocytochemistry/ Immunofluorescence abreview for Anti-alpha Adaptin [AP6] antibody

 Good
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
0.1% BSA and 1% serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 16°C
Sample
Mouse Cell (mouse embryonic stem cell)
Specification
mouse embryonic stem cell
Permeabilization
Yes - 0.1% triton x
Fixative
methanol/acetone
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Abcam user community

Verified customer

Submitted May 19 2014

Western blot abreview for Anti-alpha Adaptin antibody [AP6]

 Poor
Abreviews
abreview image
Application
Western blot
Loading amount
100000 cells
Gel Running Conditions
Reduced Denaturing (10% gel)
Sample
Mouse Cell lysate - whole cell (mouse embryonic stem cells)
Specification
mouse embryonic stem cells
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 16°C
Read More

Abcam user community

Verified customer

Submitted Jan 09 2014

Immunocytochemistry/ Immunofluorescence

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 22°C
Sample
Human Cell (HeLa)
Specification
HeLa
Permeabilization
Yes - 0.2% Triton in PBS
Fixative
Formaldehyde
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Abcam user community

Verified customer

Submitted Mar 05 2013

Question

Thank you for your reply. Since we got AP2 antibody from sigma and that antibody works, we did not really do trouble-shooting. About the refund, we always buy antibody from your company, so I am thinking that the active code that can be used at next purchase for the same price products may be good for us. Thank you.Best

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Answer

Thank you for contacting us.

Your credit note ID is XXXXX.

I am sorry that this antibody did not perform as stated on the datasheet. I have asked our accounting department to issue a credit note for you, which can be redeemed against the invoice of a future order by passing it on to your purchasing department. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. If you have questions on how to use the credit note, please contact our accounting department.

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

ReviewID: 33690 AbID: 2730, Anti-alpha Adaptin antibody [AP6] Rating: Inconclusive Image: Anti-alpha Adaptin antibody [AP6] for Western blot (Mouse) Sample: Species: Mouse Type: Cell lysate - whole cell Loading amount: 90 µg Specification: mouse neuro2A Application: Application: Gel Running Conditions: Non-reduced Non-Denaturing (Native) (8% running gel) Blocking step: Milk as blocking agent for 1 hour at 25°C Blocking Concentration: 5% Other detail: Dilution: 1/300 Incubation time: 12 hours at 4°C Secondary Antibody: Name: Non-Abcam Antibody was used: anti-mouse IgG(whole molecule)-peroxidase, antibod Conjugation: HRP Dilution: 1/5000 Detection: Detection method: ECL Exposure: 30 minutes and 0 seconds Bands: Specific: no kDa Non-specific: too many kDa Positive Control: we use sigma A4325 monoclonal anti-a-adaptin (AP2)as a control,same sample solubilization, it is able to detecting AP2 pretty well. Additional Data:

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Answer

Thank you for submitting an Abreview of ab2730. As you may have noticed, your review has now been published on our website.

Since you obtained poor results using the antibody in a tested species and application, we would like to follow up on this to see if we can possibly improve the results you are seeing with this antibody.

Based on the information in your review, I feel that altering few step in your protocol may help improve your results. I wonder if you have tried troubleshooting the protocol. If yes, could you provide details.

This antibody is covered by our Abpromise, so if the antibody was purchased within the past 6 months, I would be happy to discuss a refund or replacement with you to resolve this issue.

I look forward to your reply.

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Western blot abreview for Anti-alpha Adaptin antibody [AP6]

 Inconclusive
Abreviews
abreview image
Application
Western blot
Sample
Mouse Cell lysate - whole cell (mouse neuro2A)
Loading amount
90 µg
Specification
mouse neuro2A
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (8% running gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Read More

Abcam user community

Verified customer

Submitted Dec 21 2012

1-10 of 14 Abreviews or Q&A

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