Overview

  • Product name
  • Description
    Rabbit polyclonal to ACK1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Rat, Human
    Predicted to work with: Mouse, Cow, Dog, Rhesus monkey
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1000 to the C-terminus of Human ACK1.

    Read Abcam's proprietary immunogen policy (Peptide available as ab74487.)

  • Positive control
    • This antibody gave a positive signal in the following Whole Cell Lysates: HeLa, HepG2, PC12, SHSY-5Y, T24/83, HT 1080

Properties

Applications

Our Abpromise guarantee covers the use of ab65108 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 1 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 114 kDa).

Target

  • Function
    Non-receptor tyrosine kinase that regulates the activity of a number of proteins by tyrosine phosphorylation especially proteins critical for cell survival, cell growth, and proliferation. Activates AKT1 by phosphorylating it on 'Tyr-176' resulting in its activation. Phosphorylates AR on 'Tyr-267' and 'Tyr-363' and promotes its recruitment to the androgen-responsive enhancers (AREs). Phosphorylates WWOX on 'Tyr-287'. Downstream effector of CDC42 which mediates CDC42-dependent cell migration via phosphorylation of BCAR1. Binds to both poly- and mono-ubiquitin and regulates ligand-induced degradation of EGFR. Participates in clathrin-mediated endocytosis. May be involved both in adult synaptic function and plasticity and in brain development.
  • Tissue specificity
    The Tyr-284 phosphorylated form shows a significant increase in expression in breast cancers during the progressive stages i.e. normal to hyperplasia (ADH), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) and lymph node metastatic (LNMM) stages. It also shows a significant increase in expression in prostate cancers during the progressive stages.
  • Sequence similarities
    Belongs to the protein kinase superfamily. Tyr protein kinase family.
    Contains 1 CRIB domain.
    Contains 1 protein kinase domain.
    Contains 1 SH3 domain.
    Contains 1 UBA domain.
  • Domain
    The EBD (EGFR-binding domain) domain is necessary for interaction with EGFR.
    The SAM-like domain is necessary for NEDD4-mediated ubiquitination. Promotes membrane localization and dimerization to allow for autophosphorylation.
    The UBA domain binds both poly- and mono-ubiquitin.
  • Post-translational
    modifications
    Autophosphorylation regulates kinase activity. Phosphorylation on Tyr-518 is required for interaction with SRC.
    Ubiquitinated by NEDD4. Its EGF-induced degradation is EGFR activation-dependent and is processed by lysosomes, not proteasomes.
  • Cellular localization
    Cell membrane. Nucleus. Endosome. Cell junction > adherens junction. Cytoplasmic vesicle membrane. The Tyr-284 phosphorylated form is expressed both in the membrane and nucleus. Co-localizes with EGFR on the endosomes.
  • Information by UniProt
  • Database links
  • Alternative names
    • Acetate kinase 1 antibody
    • Acetokinase 1 antibody
    • ACK 1 antibody
    • ACK antibody
    • ACK-1 antibody
    • ACK1 antibody
    • ACK1_HUMAN antibody
    • Activated Cdc42 associated kinase 1 antibody
    • Activated CDC42 kinase 1 antibody
    • Activated p21cdc42Hs kinase antibody
    • FLJ44758 antibody
    • FLJ45547 antibody
    • p21cdc42Hs antibody
    • TNK 2 antibody
    • TNK2 antibody
    • Tyrosine kinase non receptor 2 antibody
    • Tyrosine kinase non receptor protein 2 antibody
    • Tyrosine kinase non-receptor protein 2 antibody
    see all

Images

  • All lanes : Anti-ACK1 antibody (ab65108) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
    Lane 4 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate
    Lane 5 : T24/83 (Human bladder carcinoma) Whole Cell Lysate
    Lane 6 : HT 1080 (Human fibrosarcoma) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 114 kDa
    Observed band size: 120 kDa
    why is the actual band size different from the predicted?


    Exposure time: 4 minutes
  • ICC/IF image of ab65108 stained HeLa cells. The cells were 4% PFA 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 (ab65108, 1µg/ml) 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.

References

This product has been referenced in:
  • Maxson JE  et al. Identification and Characterization of Tyrosine Kinase Nonreceptor 2 Mutations in Leukemia through Integration of Kinase Inhibitor Screening and Genomic Analysis. Cancer Res 76:127-38 (2016). Read more (PubMed: 26677978) »
  • Wilson C  et al. Overcoming EMT-associated resistance to anti-cancer drugs via Src/FAK pathway inhibition. Oncotarget 5:7328-41 (2014). WB ; Human . Read more (PubMed: 25193862) »
See all 2 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

Inquiry: Hi there, I'm trying to detect endogenous Ack1 in various cell lysates with Ab65108 by western blot but am unable to measure it. CELL LYSATES I prepare the cell lysates in RIPA buffer (Pierce cat no. 89901) supplemented with 1:100 phosphatase inhibitor cocktail 2 (Sigma cat no. P5726) and 3 (Sigma cat no. P0044) and 1:50 protease inhibitor cocktail (Sigma cat no. P8340) LOADING I load 20µg/lane of cell lysate that has been sonicated in 1X SDS loading buffer (4X NUPAGE LDS Sample Buffer (Invitrogen NP0007) + 10X NUPAGE Sample Reducing Agent (Invitrogen NP0009) TRANSFER The proteins were separated by running the gels for 55mins at constant voltage of 200V in 1X MOPS SDS Running Buffer (Invitrogen NP0001) with NuPAGE Antioxidant (Invitrogen cat no. NP0005) The proteins from the gels were transferred onto a nitrocellulose membrane (Invitrogen cat no. IB301001) for 7 mins, program 3, using the iBLOT Gel Transfer Device (Invitrogen cat no. 1B1001UK). BLOCKING I block overnight at 4oC on a rollermixer in Odyssey Block (LiCOR cat no. 927-40000) ANTIBODIES The membrane was probed for 2 hours at RT on the rollermixer with 1µg/ml of Ab65108 in TBST. Membranes were rinsed 1x and washed 3X5mins with TBST The membrane was probed for 1 hour at RT on the rollermixer with 1 in 10,000 dilution of anti-rabbit-IRdye-800CW (LiCOR cat no. 926-32211) + 0.01% SDS. Membranes were rinsed 1x and washed 3X5mins with TBST followed by rinsed 1x and washed 1X5mins with TBS I look forward to any suggestions that you may have to be able to detect endogenous Ack1 levels.

Read More
Answer

Thank you for contacting us.
I have double checked the western blots obtained for this particular lot, and the results looked good.
The lysis buffer we used to prepare our cell lysates is the following:
300 ul RIPA buffer
100 ul 10x Protease inhibitor cocktail
5 ul sodium orthovanadate solution (made from 19 mg of sodium orthovanadate powder and 500ul water)
If you are detecting ACK1 in your overexpressing tissues, then you may be having problems detecting endogenous ACK1 if the levels are very low. I have a few suggestions which may improve your results:
1. You could try performing overnight incubations at 4ºC with the primary antibody instead of 2 hours as this may help detect low levels of protein.
2. Alternatively, you could try loading more cell lysate e.g. 30ug.
3. In addition, too much blocking could lead you to not being able to visualise your protein of interest so I suggest trying a blocking step of 3% BSA for 1 hour.
If these suggestions do not improve your results, please do not hesitate to contact me again and I will be more than happy to offer you a free of charge replacement, refund or credit note. Unfortunately, if you would like a free of charge replacement, we only have the same lot as you have already tried in stock so I am reluctant to send you the same lot again as you may have the same problems. Alternatively I could suggest Anti-ACK1 antibody (ab77694) (www.abcam.com/ab77694) or Anti-ACK1 antibody - N-terminal (ab137506) (www.abcam.com/ab137506) both of which are guaranteed to work in human in western blot.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Question

Inquiry: Hi there, I'm trying to detect endogenous Ack1 in various cell lysates with Ab65108 by western blot but am unable to measure it. CELL LYSATES I prepare the cell lysates in RIPA buffer (Pierce cat no. 89901) supplemented with 1:100 phosphatase inhibitor cocktail 2 (Sigma cat no. P5726) and 3 (Sigma cat no. P0044) and 1:50 protease inhibitor cocktail (Sigma cat no. P8340) LOADING I load 20µg/lane of cell lysate that has been sonicated in 1X SDS loading buffer (4X NUPAGE LDS Sample Buffer (Invitrogen NP0007) + 10X NUPAGE Sample Reducing Agent (Invitrogen NP0009) TRANSFER The proteins were separated by running the gels for 55mins at constant voltage of 200V in 1X MOPS SDS Running Buffer (Invitrogen NP0001) with NuPAGE Antioxidant (Invitrogen cat no. NP0005) The proteins from the gels were transferred onto a nitrocellulose membrane (Invitrogen cat no. IB301001) for 7 mins, program 3, using the iBLOT Gel Transfer Device (Invitrogen cat no. 1B1001UK). BLOCKING I block overnight at 4oC on a rollermixer in Odyssey Block (LiCOR cat no. 927-40000) ANTIBODIES The membrane was probed for 2 hours at RT on the rollermixer with 1µg/ml of Ab65108 in TBST. Membranes were rinsed 1x and washed 3X5mins with TBST The membrane was probed for 1 hour at RT on the rollermixer with 1 in 10,000 dilution of anti-rabbit-IRdye-800CW (LiCOR cat no. 926-32211) + 0.01% SDS. Membranes were rinsed 1x and washed 3X5mins with TBST followed by rinsed 1x and washed 1X5mins with TBS I look forward to any suggestions that you may have to be able to detect endogenous Ack1 levels.

Read More
Answer

Thank you for taking time to submit your protocol and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.
Having reviewed the protocol details, I have no suggestions to improve your western blot protocol as it seems very good. The only comments I can add is that we blocked our membrane and diluted our antibodies in 3% BSA. We also blocked for 1 hr at room temperature and incubated with the primary antibody overnight at 4ºC which you could try but I doubt this will improve the results much.
It appears that you may have received a faulty vial. I apologize for the inconvenience and am pleased to offer you a free of charge replacement, credit note, or refund in compensation.
Unfortunately, if you would like a free of charge replacement, we only have the same lot as you have already tried in stock so I am reluctant to send you the same lot again as you may have the same problems. Alternatively I could suggest Anti-ACK1 antibody (ab77694) (www.abcam.com/ab77694) or Anti-ACK1 antibody - N-terminal (ab137506) (www.abcam.com/ab137506) both of which are guaranteed to work in human in western blot.
Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% TritonX100 in PBS

Dr. Kirk Mcmanus

Verified customer

Submitted Nov 20 2009

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