Overview

  • Product name
  • Description
    Goat polyclonal to PLK4
  • Host species
    Goat
  • Tested applications
    Suitable for: ICC/IF, Flow Cyt, WBmore details
  • Species reactivity
    Reacts with: Rat, Human
    Predicted to work with: Mouse, Cow, Dog
  • Immunogen

    Synthetic peptide:

    ATCIGEKIEDFKVGN

    , corresponding to N terminal amino acids 2-16 of Human PLK4.

  • Positive control
    • Human colon lysate
  • General notes
    GenBank Accession Number - NP_055079

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer
    Preservative: 0.02% Sodium Azide
    Constituents: 0.5% BSA, Tris buffered saline, pH 7.3
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    Purified from goat serum by ammonium sulphate precipitation followed by antigen affinity chromatography using the immunizing peptide.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab2642 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 10 µg/ml.
Flow Cyt Use a concentration of 10 µg/ml.
WB Use a concentration of 1 - 3 µg/ml. Detects a band of approximately 110 kDa (predicted molecular weight: 109 kDa).Can be blocked with Human PLK4 peptide (ab22924).

Target

  • Function
    Serine/threonine-protein kinase that plays a central role in centriole duplication. Able to trigger procentriole formation on the surface of the parental centriole cylinder, leading to the recruitment of centriole biogenesis proteins such as SASS6, CENPJ/CPAP, CP110, CEP135 and gamma-tubulin. When overexpressed, it is able to induce centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole during S phase. Its central role in centriole replication suggests a posible role in tumorigenesis, centrosome aberrations being frequently observed in tumors. Also involved in trophoblast differentiation by phosphorylating HAND1, leading to disrupt the interaction between HAND1 and MDFIC and activate HAND1. Phosphorylates CDC25C and CHEK2/CHK2.
  • Sequence similarities
    Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. CDC5/Polo subfamily.
    Contains 1 POLO box domain.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Ubiquitinated; leading to its degradation by the proteasome.
    Tyrosine-phosphorylated by TEC.
  • Cellular localization
    Cytoplasm > cytoskeleton > centrosome > centriole. Nucleus > nucleolus. Cleavage furrow. Associates with centrioles throughout the cell cycle. According to PubMed:16244668, it is not present at cleavage furrows.
  • Information by UniProt
  • Database links
  • Alternative names
    • PLK 4 antibody
    • PLK-4 antibody
    • PLK4 antibody
    • PLK4_HUMAN antibody
    • Polo like Kinase 4 antibody
    • Polo-like kinase 4 antibody
    • Protein serine/threonine kinase antibody
    • SAK antibody
    • Serine/Threonine Kinase 18 antibody
    • Serine/threonine kinase antibody
    • Serine/threonine protein kinase 18 antibody
    • Serine/threonine protein kinase PLK4 antibody
    • Serine/threonine protein kinase Sak antibody
    • Serine/threonine-protein kinase 18 antibody
    • Serine/threonine-protein kinase PLK4 antibody
    • Serine/threonine-protein kinase SAK antibody
    • Snk Akin Kinase antibody
    • STK 18 antibody
    • STK18 antibody
    see all

Images

  • Immunofluorescence analysis of paraformaldehyde fixed HeLa cells staining PLK4. Cells were permeabilized with 0.15% Triton. Samples were incubated with primary anitbody for 1 hour at 10µg/ml. An Alexa Fluor 488 was used as the secondary antibody. DAPI was used as a nuclear counterstain. Unimmunized goat IgG (10µg/ml) was used as the negative control.
  • Immunofluorescence analysis of paraformaldehyde fixed A431 cells staining PLK4. Cells were permeabilized with 0.15% Triton. Samples were incubated with primary anitbody for 1 hour at 10µg/ml. An Alexa Fluor 488 was used as the secondary antibody. DAPI was used as a nuclear counterstain. Unimmunized goat IgG (10µg/ml) was used as the negative control.
  • Anti-PLK4 antibody (ab2642) at 2 µg/ml + human colon lysate in RIPA buffer at 35 µg

    Developed using the ECL technique.

    Predicted band size: 109 kDa
    Observed band size: 110 kDa
    why is the actual band size different from the predicted?


    Exposure time: 1 hour

References

This product has been referenced in:
  • Czub B  et al. TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene. PLoS One 11:e0148678 (2016). Read more (PubMed: 26872363) »
  • Rosario CO  et al. A novel role for Plk4 in regulating cell spreading and motility. Oncogene N/A:N/A (2014). Read more (PubMed: 25174401) »
See all 5 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Answer

I have organised a new vial to be sent to you, you should get a confirmation e-mail for this "order" soon. Please could you let me know how you get on with this batch? I would very much appreciate your feedback.

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Answer

The antibody in stock is a new batch and therefore has not been tested by other customers. I have arranged a credit note to be issued to you. This antibody sells well and the reported problems are therefore few. I wish you all the best with your research,

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Question

BATCH NUMBER 58237 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal at all SAMPLE 3T3 cells expressing recombinant flag-tagged human plk4 PRIMARY ANTIBODY 3 ug/mL anti plk-4, in 1% BSA tris-saline, 0.05% tween SECONDARY ANTIBODY 1:5000 anti-goat-hrp (santa cruz) DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED POSITIVE CONTROL: recombinant flag-tagged human plk4 was expressed in 3T3 cells and immunoprecipitated with anti-flag antibody. Membranes were probed with anti-flag antibody and a clear band of ~120 kDa was observed. When a parallel membrane was probed with anti-plk4, there is no band detected at all. NEGATIVE: 3T3 cells were transfected with empty vector (FLAG-CMV 7.0, sigma) and immunoprecipitated with anti-flag. When probed with anti-flag, there is no band at 120 kDa. ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION Lysed at 4 degrees with protease and phosphatase inhibitors. 500 ug of protein immunoprecipitated with 2 ug of anti-flag antibody AMOUNT OF PROTEIN LOADED All of IP ELECTROPHORESIS/GEL CONDITIONS reducing 10% sds-page TRANSFER AND BLOCKING CONDITIONS semi-dry tris-glycine methanol HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES This antibody does not recognize plk-4 at all. I have tried this numerous times with whole cell lysate and recombinant proteins. I have wasted time and money and am very unhappy with tech support thus far. Please either send me a full refund or a new lot of the antibody that has actually been tested and is known to work.

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Answer

I'm terribly sorry you are having problems with ab2642. We have a new batch in stock which I could send to you or I can arrange a refund or credit note. Please let me know which option you would prefer and accept my apologies for this problem, I look forward to hearing from you,

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Question

I am very happy to get your response. And following protocols were my detailed Western blotting analysis. ANTIBODY CODE ab2642 DESCRIPTION OF THE PROBLEM high background, the major band is the wrong size (66 KDa)?besides human placenta lysate that is at appropriate 50 KDa SAMPLE ?1?positive control as your datasheet using human placenta lysate? 2?various cancer cell lines including HeLa?PC3?HBL100?Au565?MCF7?A431 PRIMARY ANTIBODY incubated with anti-Plk4 antibody (1?2000) for overnight at 4? dissolving 0.3g BSA/10ml TBST buffer. Washed 4 times for 8 minutes using TBST buffer. SECONDARY ANTIBODY incubated with anti-goat conjugated-HRP antibody(1? 3000) for 40 minutes at room-temperature dissolving 0.3g milk/10ml TBST buffer. Washed 4 times for 6 minutes using TBST buffer. DETECTION METHOD used ECL detection system (Amersham) ANTIBODY STORAGE CONDITIONS Aliquot and store at -20? SAMPLE PREPARATION Cell lines and placenta tissue lysed with lysis buffer. And then freeze-thaw 3 times. Centrifuge at 15000 rpm for 10 minutes. The supernatant was subjected to the BCA Quantitation assay(BioRed).?lysis buffer?50mM Hepes-KOH pH7.4, 150mM NaCl, 1?TritonX-100, protease inhibitor ? AMOUNT OF PROTEIN LOADED 30 ug or 50 ug ELECTROPHORESIS/GEL CONDITIONS 7.5% SDS-PAGE gel TRANSFER AND BLOCKING CONDITIONS Transferred to PVDF membrane(MILLIPORE) in a trans-cassette. Blocked with 5?milk/TBST buffer at room-temperature for 1 hour. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No, but I have used two different secondary antibodys from distinct sources. DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Almost the same Above is my experimental protocol. Hope that you can find the root of the problem though these described information. Please help and contact me again as soon as possible. I'm looking forward to your next message.

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Answer

Thank you for the information. I have now a clearer idea of your problem and would like to help by suggesting some modifications to your protocol. I think the problem might be a degradation of your protein before it is recognised by the antibody as you mention freeze thawing it several times and I would like to recommend another lysis buffer (RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton -100, 1% Sodium deoxycholate, 0.1% SDS). Following homogenisation over 20 minutes on ice centrifuge at 13,000 rpm for 5 min in a centrifuge (which is pre-cooled and stays cool)transfer the supernatant into clean tubes and do the protein concentration as usual. Add an equal volume of 2 x SDS sample buffer (made with SDS and betamercaptoethanol) and boil for 5 minutes. You can then store the lysates at -80C until use. I think the rest of your WB protocol looks fine. To minimise the background you report I would suggest blocking your membrane in non-fat milk (3-5%w/v in TBST, filtered) for one hour and not incubating the other antibodies in blocking buffer (just TBST). After blocking, rinse once (for 10sec)the membrane with TBST.This will remove excess milk which can cause sometimes high background. It could also be due to too much secondary antibody or simply that the secondary has gone off...have you tried incubating with no primary, this will help determining if the problem comes from the secondary. Try also the secondary in other WBs for the proteins and see if it gives you problems. Also, wash extensively the membrane between steps, it's always better to wash many times for short periods of time (4-5min) than a few long washes. I hope this information helps, please do not hesitate to contact me again if you still have problems, Good luck!

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Answer

Thank you for your e-mail. Please find enclosed the protocol. Tissue Lysis: Tissue chunks were weighed and cut into approx 1mm cubes using a razor blade. The tissue was transferred to a handheld homogenizer and 3 ml of ice-cold RIPA buffer was added per 1g of tissue. The tissue was gently homogenised over 20 minutes on ice. The resulting lysate was aliquotted into 1.5 ml microfuge tubes and centrifuged at 13,000 rpm for 5 min in a microfuge. The supernatant was transferred into clean tubes and its protein concentration was measured with BioRad protein assay. The concentration was then adjusted to 5 mg/ml with RIPA lysis buffer. An equal volume of 2 x SDS sample buffer was added and the cell lysate was boiled for 5 minutes. Lysates were stored at -80C until use.(RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton -100, 1% Sodium deoxycholate, 0.1% SDS). We hope this will help.

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Question

ANTIBODY CODE ab2642 DESCRIPTION OF THE PROBLEM High background, major band is the wrong size (72 kDa) SAMPLE Multiple samples used: 1) Cell line BJ- human fibroblast line using exponentially growing cells and nocodazole treated cells 2) Human colorectal cancer tissue lysates 3) Human colorectal cancer cell lines PRIMARY ANTIBODY Incubated with 3ug/mL of your anti-plk4 antibody overnight at 4 degrees celcius in 1%BSA Tris/Saline + 0.05% Tween. Washed 3x for 5 minutes each with Tris/Saline with 0.05% Tween. SECONDARY ANTIBODY Used 1:2000 santa-cruz donkey anti-goat HRP conjugated antibody with 1%BSA Tris/Saline +0.05% Tween. Washed 3X for 5 minutes each with Tris/Saline with 0.05% Tween. DETECTION METHOD used amersham chemiluminescent detection system, femto grade POSITIVE AND NEGATIVE CONTROLS USED Positve control: M phase enriched cells (blocked 48 hrs with nocodazole) Negative control: G0 phase enriched cells (Blocked 48 hrs with serum starvation) ANTIBODY STORAGE CONDITIONS Aliquoted and stored at -20 SAMPLE PREPARATION Cell lines and tissues lysed in TNT with complete protease inhibitors (Roche) plus phosphatase inhibitors. Diluted with SDS-PAGE loading dye with 2-mercaptoethanol. Boiled at 100 degrees celcius for 10 minutes. AMOUNT OF PROTEIN LOADED 30 ug. ELECTROPHORESIS/GEL CONDITIONS Reducing 8% SDS-PAGE gel TRANSFER AND BLOCKING CONDITIONS Transferred to immbolion-p PDVF membranes using tris/glycine/methanol buffer in a mini-trans blot (BioRad) system. Blocked by drying in fume hood overnight. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No ADDITIONAL NOTES I just can't seem to get a major band that is greater than 100 kDa... and my blots are very dirty. Please help!

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Answer

Thank you for your enquiry and for forwarding your detailed protocol. This antibody has been tested for Western blot application using Human Placenta Lysate. We would strongly suggest running a positive control along with the samples. Also you need to run "no primary" control to see if the secondary antibody gives a clear background. You have mentioned in your e-mail that "Blocked by drying in fume hood overnight" - we are very sorry but haven't heard of anyone doing this before. You may wish to try the following: after protein transfer on PVDF membrane, block the membrane in 2.5% skimmed milk in TBS-T for 1 hr at room temperature with agitation. Note that if PVDF membranes dry out after transfer therefore they should be "rewetted" with 100% methanol – there should be some information about this in the membrane product literature. We hope this will help. Should you still need more technical support, please do not hesitate to contact us again.

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