Overview

  • Product name
    Anti-IRAK-1 antibody - BSA and Azide free
    See all IRAK-1 primary antibodies
  • Description
    Rabbit polyclonal to IRAK-1 - BSA and Azide free
  • Host species
    Rabbit
  • Specificity
    No cross reactivity to IRAK-2.
  • Tested applications
    Suitable for: WB, IP, IHC-P, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Rhesus monkey
  • Immunogen

    Synthetic peptide corresponding to Human IRAK-1 aa 700-712 (C terminal).
    (Peptide available as ab6230)

  • Positive control
    • THP-1 or HeLa whole cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab238 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
WB 1/1000. Predicted molecular weight: 80 kDa.Can be blocked with IRAK-1 peptide (ab6230). (THP-1 or HeLa whole cell) from non-activated cells.
IP Use a concentration of 5 µg/ml.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

 

ICC/IF Use a concentration of 10 µg/ml.

Target

  • Function
    Binds to the IL-1 type I receptor following IL-1 engagement, triggering intracellular signaling cascades leading to transcriptional up-regulation and mRNA stabilization. Isoform 1 binds rapidly but is then degraded allowing isoform 2 to mediate a slower, more sustained response to the cytokine. Isoform 2 is inactive suggesting that the kinase activity of this enzyme is not required for IL-1 signaling. Once phosphorylated, IRAK1 recruits the adapter protein PELI1.
  • Tissue specificity
    Isoform 1 and isoform 2 are ubiquitously expressed in all tissues examined, with isoform 1 being more strongly expressed than isoform 2.
  • Sequence similarities
    Belongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family. Pelle subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Autophosphorylated or is transphosphorylated by IRAK4 following recruitment to the IL-1RI. In the case of isoform 1, this is linked to ubiquitination and degradation.
    Polyubiquitinated; after cell stimulation with IL-1-beta. Polyubiquitination occurs with polyubiquitin chains linked through 'Lys-63'.
  • Information by UniProt
  • Database links
  • Alternative names
    • AA48924 antibody
    • Il1rak antibody
    • Interleukin 1 receptor associated kinase 1 antibody
    • Interleukin-1 receptor-associated kinase 1 antibody
    • IRAK antibody
    • IRAK-1 antibody
    • Irak1 antibody
    • IRAK1-S antibody
    • IRAK1_HUMAN antibody
    • mPLK antibody
    • OTTHUMP00000026014 antibody
    • OTTHUMP00000026015 antibody
    • OTTHUMP00000026020 antibody
    • OTTHUMP00000180621 antibody
    • Pelle antibody
    • Pelle homolog antibody
    • Pelle-like protein kinase antibody
    • Plpk antibody
    see all

Images

  • All lanes : Anti-IRAK-1 antibody - BSA and Azide free (ab238) at 1/2000 dilution

    Lane 1 : THP-1 (THP)whole cell lysate
    Lane 2 : HeLa (HL) whole cell lysate

    Predicted band size: 80 kDa
    Observed band size: 80 kDa

  • ab238 at 10µg/ml staining Hela cells by ICC/IF
  • IHC image of ab238 staining in human breast carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab238, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • ab238 staining IRAK-1 in Human platelet cells by Flow cytometry.
    Cells were fixed in paraformaldehyde and permeabilized using 0.1% Triton-X-100 in 2% BSA for 15 minutes. Primary antibody used at a 1/200 dilution and incubated for 16 hours at 4°C. The secondary antibody used was an Alexa Fluor®488 conjugated chicken anti-rabbit IgG (H+L) at a 1/500 dilution.

    P : Permeabilized US : Unstained (Red Peak) IGG RB : IgG Rabbit (Blue Peak) IRAK Ab (Green Peak)

    See Abreview

  • ab238 staining IRAK-1 in murine RAW 264.7 cells by Immunocytochemistry/ Immunofluorescence.Cells were fixed in paraformaldehyde, permeabilized using 0.1% Triton-X100 in 2% BSA for 15 minutes, blocked with 2% BSA for 1 hour at 22°C and then incubated with ab238 at a 1/150 dilution for 16 hours at 4°C. The secondary used was an Alexa-Fluor 488 conjugated chicken anti-rabbit IgG (H+L) used at a 1/1000 dilution.

    See Abreview

  • IRAK was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to IRAK-1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab238.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 80kDa: IRAK-1

  • Immunofluorescence of IRAK-1 in HeLa cells using ab238 at 20 ug/ml.

References

This product has been referenced in:
  • Chen Y  et al. MicroRNA-146a-5p attenuates irradiation-induced and LPS-induced hepatic stellate cell activation and hepatocyte apoptosis through inhibition of TLR4 pathway. Cell Death Dis 9:22 (2018). WB ; Mouse . Read more (PubMed: 29348414) »
  • Wang Z  et al. Chronic constriction injury-induced microRNA-146a-5p alleviates neuropathic pain through suppression of IRAK1/TRAF6 signaling pathway. J Neuroinflammation 15:179 (2018). WB, IHC (PFA fixed) ; Rat . Read more (PubMed: 29885668) »
See all 8 Publications for this product

Customer reviews and Q&As

1-10 of 21 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Rhesus monkey Cell (Colon)
Specification
Colon
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton-x-100
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 21 2013

Question
Answer

I have checked your new order 1217270 and it looks that both antibodies (abhttp://ops.adminsite.com/admin/data/fs_ab.cfm?&intAbID=119289 and abhttp://ops.adminsite.com/admin/data/fs_ab.cfm?&intAbID=135027) are out of stock at the moment. We have reordered the items but it may take a week to get them. According to our record, we should be able to get them by 18th December.

Could you please drop me an e-mail to confirm if you are happy to wait.

I apologize for any inconvenience caused and look forward to hearing from you soon.

Read More

Answer

Thank you for getting back to me.

This is to confirm that I have placed a new order for you - for one vial of ab238 and another vial of ab64171 as free of charge replacements and the new order number is 1217226.

I would kindly advise you to run a non-stimulated positive control cell line which expresses the target protein endogenously and to apply lysis buffer (i.e. RIPA) which contains a mixture of different proteinase inhibitors to prevent the degradation of the protein of interest.

Recommended positive controls:

ab238 - THP-1 or HeLa whole cell lysate;

ab64171- Jurkat whole cell lysate

I hope the second vials will perform better, and please do let me know how you are getting on with these two products.

Good luck!

Read More

Answer

Thank you for getting back to me and for passing some useful details. Your co-operation is much appreciated.

As our Abpromise indicates, 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.

I can certainly offer replacement vials to you if you wish to carry on with the experiments.

Could you please confirm the Abcam Order Number or you Purchase Order Number and the date of purchase.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

According to Swiss-Prot database, IRAK has 4 different isoforms (P51617-1, P51617-2, P51617-3, P51617-4) and these alternative splice variants have different sizes. You can find some further details at this site:

http://www.uniprot.org/uniprot/P51617

One more comment that RT-PCR data should be treated carefully since, there is no clear correlation between mRNA and protein levels and sometimes it is not easy to predict the protein expression levels from quantitative mRNA data.

There are several publications available which delineate the technical boundaries of the approaches for quantitative analysis of protein expression and reveal that simple deduction from mRNA transcript analysis is insufficient.

Please do let me know how you wish to proceed with your enquiry. I look forward to hearing from you soon.

Read More

Answer

Thank you for getting back to me.

I can find the 3 pdf files attached to your response but it seems that the ppt (Powerpoint) file did not come through.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

1) Control for siRNA experiments:

Have you done a time-course experiment with siRNA treated cells to select the most optimal time point for the studies?
How have you selected the sequence of siRNA? Have you tried more than one SiRNA sequence? Could you check if the sequence corresponds correctly to IRAK and CARD10 protein?
Have you checked the transfection efficiency and optimized the length of time for knockout to take effect?
Have you checked the transfection by fluorescence tags and the knockout by RT-PCR?
Have you used the correct endogenous positive and negative controls? Have you applied scrambled siRNA or standard negative siRNA controls?



2) RIPA buffer:

Laemmli buffer does not contain any proteinase inhibitors to protect the target protein form degradation. I would suggest using RIPA buffer and you can find the composition of the this solution at this site:

https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1



3) Positive control:

Since the cells are stimulated with IFN gamma, this may modify/alter the expression pattern of the target. It would be worth running good positive control alongside with the samples. It would be also be worth checking the cell viability after treatment.

ab238 - THP-1 or HeLa whole cell lysate;

ab64171- Jurkat whole cell lysate

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Read More

Answer

I am sorry to hear that you have been experiencing problems using these two products (ab238 and ab64171) in the application that you wish. Thank you very much for sending the WB images.

In order to assess the quality of our products I would ask that you complete a brief questionnaire relating to the application used. Often it is possible to make suggestions that may help resolve problems experienced using a particular product.

As our Abpromise indicates, 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.

All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience.

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

I am particularly interested in the following information:

- lysis buffer used,

- positive control,

- summary of the siRNA experiment (sequence for siRNA, transfection efficiency, endogenous positive and negative control)

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

Answer

Thank you for your inquiry.

I can confirm that the protocol for flow cytometry that we know of for this antibody includes an incubation time of 16 hours at 4C.

Please feel free to use your standard protocol if you find that this is working too for this antibody. In case the staining is week I can suggest too prolong the incubation time. A longer incubation will achieve an optimal saturation.

If in your hand a shorter incubation time is working well, i would like to encourage you to also write an Abreview. Other researchers will value this information. And you would earn Abpoints that can be redeemed as discount on future purchases or as Amazon vouchers.

I hope this information is helpful and wish you good luck with your research.

Read More

Answer

I am sorry to hear that the result did not improve.

To proceed further in this case could you let me know, how the antibody was stored and what was the purchase order number?

Thank very much for your cooperation in this case!

Read More

Answer

Thank you for submitting an Abreview of ab238. 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 the step in protocol may help toimprove your results. Did you try any troubleshooting of protocol?

Additionally, 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.

Read More
Abreviews
Application
Western blot
Sample
Mouse Cell lysate - whole cell (RAW macrophages cell line)
Loading amount
50 µg
Specification
RAW macrophages cell line
Treatment
LPS and peptidoglycan
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. Varkha Agrawal

Verified customer

Submitted Sep 11 2012

1-10 of 21 Abreviews or Q&A

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