Overview

  • Product name
    Anti-KAP1 antibody [EPR5249]
    See all KAP1 primary antibodies
  • Description
    Rabbit monoclonal [EPR5249] to KAP1
  • Host species
    Rabbit
  • Specificity
    Although the immunogen is from a phosphor-peptide, this antibody detects phospho and non-phospho KAP1. Based on a peptide blocking experiment it has been found that the signal generated after non-phospho peptide blocking became much weaker, thus indicating that ab109545 shows cross-reactivity with the non-phospho KAP1 at high level.
  • Tested applications
    Suitable for: Flow Cyt, WB, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human KAP1. A phospho specific peptide corresponding to residues surrounding Serine 473 of human KAP1 was used as the immunogen.

  • Positive control
    • WB: A431 and MCF7 cell lysates IHC-P: Human colon and kidney tissue ICC/IF: Hela cells
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab109545 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
Flow Cyt 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB 1/50000 - 1/20000. Predicted molecular weight: 89 kDa.
IHC-P 1/100 - 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol. Heat up to 98 degrees C, below boiling, and then let cool for 10-20 min.
ICC/IF 1/100 - 1/250.

Target

  • Function
    Nuclear corepressor for KRAB domain-containing zinc finger proteins (KRAB-ZFPs). Mediates gene silencing by recruiting CHD3, a subunit of the nucleosome remodeling and deacetylation (NuRD) complex, and SETDB1 (which specifically methylates histone H3 at 'Lys-9' (H3K9me)) to the promoter regions of KRAB target genes. Enhances transcriptional repression by coordinating the increase in H3K9me, the decrease in histone H3 'Lys-9 and 'Lys-14' acetylation (H3K9ac and H3K14ac, respectively) and the disposition of HP1 proteins to silence gene expression. Recruitment of SETDB1 induces heterochromatinization. May play a role as a coactivator for CEBPB and NR3C1 in the transcriptional activation of ORM1. Also corepressor for ERBB4. Inhibits E2F1 activity by stimulating E2F1-HDAC1 complex formation and inhibiting E2F1 acetylation. May serve as a partial backup to prevent E2F1-mediated apoptosis in the absence of RB1. Important regulator of CDKN1A/p21(CIP1). Has E3 SUMO-protein ligase activity toward itself via its PHD-type zinc finger.
  • Tissue specificity
    Expressed in all tissues tested including spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes.
  • Pathway
    Protein modification; protein sumoylation.
  • Sequence similarities
    Belongs to the TRIM/RBCC family.
    Contains 2 B box-type zinc fingers.
    Contains 1 bromo domain.
    Contains 1 PHD-type zinc finger.
    Contains 1 RING-type zinc finger.
  • Domain
    The HP1 box is both necessary and sufficient for HP1 binding.
    The PHD-type zinc finger enhances CEBPB transcriptional activity. The PHD-type zinc finger, the HP1 box and the bromo domain, function together to assemble the machinery required for repression of KRAB domain-containing proteins. Acts as an intramolecular SUMO E3 ligase for autosumoylation of bromodomain.
    The RING-finger-B Box-coiled-coil/tripartite motif (RBCC/TRIM motif) is required for interaction with the KRAB domain of KRAB-zinc finger proteins. Binds four zinc ions per molecule. The RING finger and the N-terminal of the leucine zipper alpha helical coiled-coil region of RBCC are required for oligomerization.
    Contains one Pro-Xaa-Val-Xaa-Leu (PxVxL) motif, which is required for interaction with chromoshadow domains. This motif requires additional residues -7, -6, +4 and +5 of the central Val which contact the chromoshadow domain.
  • Post-translational
    modifications
    Phosphorylated upon DNA damage, probably by ATM or ATR. ATM-induced phosphorylation on Ser-824 represses sumoylation leading to the de-repression of expression of a subset of genes involved in cell cycle control and apoptosis in response to genotoxic stress. Dephosphorylation by the phosphatases, PPP1CA and PP1CB forms, allows sumoylation and expression of TRIM28 target genes.
    Sumoylation/desumoylation events regulate TRIM28-mediated transcriptional repression. Sumoylation is required for interaction with CHD3 and SETDB1 and the corepressor activity. Represses and is repressed by Ser-824 phosphorylation. Enhances the TRIM28 corepressor activity, inhibiting transcriptional activity of a number of genes including GADD45A and CDKN1A/p21. Lys-554, Lys-779 and Lys-804 are the major sites of sumoylation. In response to Dox-induced DNA damage, enhanced phosphorylation on Ser-824 prevents sumoylation and allows de-repression of CDKN1A/p21.
  • Cellular localization
    Nucleus. Associated with centromeric heterochromatin during cell differentiation through CBX1.
  • Information by UniProt
  • Database links
  • Alternative names
    • E3 SUMO protein ligase TRIM28 antibody
    • E3 SUMO-protein ligase TRIM28 antibody
    • FLJ29029 antibody
    • KAP 1 antibody
    • KAP-1 antibody
    • KRAB associated protein 1 antibody
    • KRAB interacting protein 1 antibody
    • KRAB-associated protein 1 antibody
    • KRAB-interacting protein 1 antibody
    • KRIP 1 antibody
    • KRIP-1 antibody
    • KRIP1 antibody
    • Nuclear corepressor KAP 1 antibody
    • Nuclear corepressor KAP-1 antibody
    • RING finger protein 96 antibody
    • RNF96 antibody
    • TF1B antibody
    • TIF1 beta antibody
    • TIF1-beta antibody
    • TIF1B antibody
    • TIF1B_HUMAN antibody
    • Transcription intermediary factor 1 beta antibody
    • Transcription intermediary factor 1-beta antibody
    • Trim28 antibody
    • Tripartite motif containing 28 antibody
    • tripartite motif containing protein 28 antibody
    • Tripartite motif-containing protein 28 antibody
    see all

Images

  • Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: HAP1 + DMSO whole cell lysate (20 µg)
    Lane 3: HAP1 + Blaomycin whole cell lysate (20 µg)
    Lane 4: TRIM28 knockout HAP1 whole cell lysate (20 µg)
    Lane 5: TRIM28 knockout HAP1 + DMSO whole cell lysate (20 µg)
    Lane 6: TRIM28 knockout HAP1 + Blaomycin whole cell lysate (20 µg)
    Lane 7: HeLa + DMSO whole cell lysate (20 µg)
    Lane 8: HeLa + Blaomycin whole cell lysate (20 µg)

    Lanes 1 - 8: Merged signal (red and green). Green - ab109545 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab109545 was shown to specifically react with KAP1 in wild type cells as signal was lost in KAP1 knockout cells. Wild-type and KAP1 knockout samples were subjected to SDS-PAGE. ab109545 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • All lanes : Anti-KAP1 antibody [EPR5249] (ab109545) at 1/50000 dilution

    Lane 1 : A431 cell lysate
    Lane 2 : A431 cell lysate treated with Lambda Phosphatase
    Lane 3 : MCF7 cell lysate
    Lane 4 : MCF7 cell lysate treated with Lambda Phosphatase

    Lysates/proteins at 10 µg per lane.

    Predicted band size: 89 kDa

  • ab109545, at a 1/100 dilution, staining KAP1 in formalin-fixed, paraffin-embedded Human colon tissue.
  • ab109545, at a 1/100 dilution, staining KAP1 in formalin-fixed, paraffin-embedded Human kidney tissue.
  • ab109545, at a 1/100 dilution, staining KAP1 in HeLa cells
  • Overlay histogram showing HeLa cells stained with ab109545 (red line). The cells were fixed with 80% methanol (5 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 (ab190545, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

References

ab109545 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Question
Answer

The IgG concentration of lot GR564755 (including GR564755-8) is 0.166 mg/ml.

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Answer

Thank you for contacting us. No, these antibodies have not been tested with overexpression or knockdown cells.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question
Answer

Thank you for your kind reminder.

As I have mentioned in my previous e-mail (sent Thu 21 Jun 12 ·11:32) we could offer some alternative antibodies for you. If you can use phospho specific anti KAP1 antibody other than S473 then we can certainly offer any of these to try ab133225 or ab70369 or ab84077.

If you wish otherwise, I could arrange a full refund for you.

Could you please get back to me and let me know how you wish to proceed.

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Answer

Thank you for your response and patience.

I have been discussing this enquiry with my colleagues in the lab. Could you please confirm if you have tried our positive control treatment, Lambda Phosphatase?

I understand that radiation also works based on the literature. I attached a dot blot to show phospho specificity. “N-P” stands for the control peptide and “P” for the phospho peptide. Please let me know if you have any questions.

I hope this helps and if I can assist further, please do not hesitate to contact me.

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Question

We have recently bought (through the local distributor) the Anti-KAP1 (phospho S473) antibody ab109545 LOT: GR54755-3 that when tested on western blots of lysates from human cells treated or untreated with radiation shows a band of the correct MW but whose levels do not change with treatment (see attached). We believe that the antibody in NOT recognizing S473 phosphorylation, but only total KAP1.
According to previously published data, KAP1 S473 phosphorylation occurs only after treatment of cells with genotoxic stress, such as ionizing radiation ( see http://www.ncbi.nlm.nih.gov/pubmed/22491012 Lee DH, et al, EMBO J. 2012 Apr 10. doi: 10.1038/emboj.2012.86. )
I would appreciate if you could send a replacement antibody of a different lot and after you checked it works as expected.

Please find attached the completed form.

I draw your attention on our longstanding technical expertise in the analysis of phosphorylated proteins from DNA damage cells (see papers listed below).


1) Abcam product code ab: 109545

2) Abcam order reference number or product batch number: GR54755-3

3) Description of the problem:

Antibody not recognizing KAP1- S473 phosphorylation after radiation treatment

4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysates from MCF7 cells

Lysis buffer: 4% SDS + 125mM Tris HCl

Protease inhibitors: PMSF

Phosphatase inhibitors: Na3Vo4

Reducing agent: β-mercaptoethanol

Boiling for ≥5 min? yes/no: Yes

Protein loaded ug/lane or cells/lane: 40µg/lane

Positive control: irradiated MCF7 cells

Negative control: untreated shCHK2 MCF7 cells

5) Percentage of gel: 4-12 % Bis-Tris gel from Invitrogen

Type of membrane: PVDF

Protein transfer verified: YES

Blocking agent and concentration: 4% Milk in 1XPBS+Tween(0.1%)

Blocking time: 1 hour

Blocking temperature: Room Temperature

6) Primary antibody (If more than one was used, describe ifn “additional notes”) :

Concentration or dilution: 1:50000

Diluent buffer : 5% BSA in TBS+Tween 0.1%

Incubation time: overnight

Incubation temperature: 15ºC

7) Secondary antibody: Donkey Anti-Rabbit IgG ECL antibody

Species Donkey

Reacts against: Rabbit

Concentration or dilution: 1:2000

Diluent buffer : 4% Milk in 1XPBS+Tween 0.1%

Incubation time: 1 Hour

Incubation temperature: 15ºC

Fluorochrome or enzyme conjugate: HRP

8) Washing after primary and secondary antibodies Buffer: 1XPBS Tween (0.1%)

Number of washes: 3 washes, 10 minutes each

9) Detection method: ECL

10) How many times have you run this staining? 3 Times

Do you obtain the same results every time? YES

What steps have you altered to try and optimize the use of this antibody? Cell lines, antibody dilution and treatments with different DNA damaging agents

Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.

Best regards

Read More
Answer

Thank you for your enquiry regarding ab109545 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

The protocol looks fine to me. There is only one thing I would like to comment on is the blocking:

For detection of phospho protein, we would suggest using 3% BSA solution - as blocking agent - rather than milk. Milk may lead to significant decrease or loss of signal intensity due to reaction of primary antibody with blocking/diluting agent.

Have you used this antibody before? Could you please confirm it?

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

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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 (Hela cells)
Loading amount
50 µg
Specification
Hela cells
Treatment
non treated, AZD7762 for 4 hrs, etoposide 2µM for 3 hrs, 50 nM AZD7762 for 1h+ etoposide 3h
Gel Running Conditions
Reduced Denaturing (8%)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 18°C

Abcam user community

Verified customer

Submitted May 15 2012

Answer

Thank you for your enquiry regarding ab109545 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody and do not get the expected results.

I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

Could you provide some further details of the protocol used and complete the following form (attached as a word document).

I am particularly interested in the followings:

- Sample preparation:

- Lysis buffer:

- Blocking agent used:

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

Read More

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