VeriBlot for IP secondary antibody (HRP) (ab131366)

Overview

  • Product nameVeriBlot for IP secondary antibody (HRP)See all IgG secondary antibodies ...
  • SpecificityVeriBlot for IP secondary antibodies are immunoblotting reagents that enable the trouble-free detection of immunoblotted target protein bands, without interference from denatured IgG. This allows to detect the (co-)immunoprecipitated protein without masking by the IgG heavy (50 kDa) and light chains (25 kDa). In general this interference tends to originate from secondary antibodies which recognize primary antibodies released with the antigen during the immunoprecipitation procedure or endogenous IgGs from the lysate itself. VeriBlot for IP secondary antibodies only recognize native (non-reduced) antibodies and therefore the detection of heavy and light chains is highly minimized, if the immunoprecipitate is fully reduced.
  • Tested applicationsWBmore details
  • ConjugationHRP

Properties

  • FormLiquid
  • Storage instructionsStore at +4°C.
  • Concentration information loading...
  • Clonality Monoclonal
  • IsotypeIgG
  • General notes

    Number of blots:
    At least 20
    (based on a 1:200 dilution in 5 ml milk). 

    Important protocol notes:
    1. The VeriBlot for IP secondary antibody (HRP) detects the following IgG polyclonal and monoclonal antibodies:

     

    SpeciesMonoclonal Isotype(s)
    BovineIgG2
    GoatIgG2
    HumanIgG1, IgG2, IgG4
    MouseIgG2a, IgG2b, IgG3
    RatIgG2C
    RabbitTotal IgG
    SheepIgG2


    2. The VeriBlot for IP secondary antibody (HRP) preferentially detects the non-reduced form over the reduced, SDS-denatured forms.

    3. IP sample should be completely reduced/denatured before loaded onto a western blot.

    4. Milk should be used as the blocking protein for the immunoblot.

    Western blot and IP resources:
    a) Western blot a beginner's guide
    b) IP protocol
    c) IP troubleshooting tips

  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab131366 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 1/40 - 1/4000. The dilution will depend on the sensitivity of the HRP substrate. The dilution range recommended is 1:40 - 1:4000. Based on a 1:200 dilution (25 µL) in 5 ml milk researchers can perform 20 western blots. This product is recommended for the western blot detection of IP samples.

VeriBlot for IP secondary antibody (HRP) images

  • ab128874 Immunoprecipitating Brd4 in human HEK293 whole cell lysate. 1000µg of cell lysate was incubated with primary antibody (1µg/mg in 50 mM Tris) and matrix (Protein G) for 16 hours at 4°C. For western blotting a HRP-conjugated Veriblot for IP secondary antibody (ab131366) (1/10000) was used to confirm successful immunoprecipation.

    See Abreview

  • ab32371 immunoprecipitating Bak in human HCT116 p53-/- whole cell lysate. 100µg of cell lysate was incubated with primary antibody (1/100) and matrix (Protein A/G) for 4 hours at 4°C. For western blotting a HRP-conjugated Veriblot for IP secondary antibody (ab131366) (1/2000) was used to confirm successful immunoprecipation.

    See Abreview

  • ab6148 Immunoprecipitating IRAK2 in human HEK293 whole cell lysate. 1000µg of cell lysate was incubated with primary antibody (1 µg/mg) and matrix (Protein G) for 16 hours at 4°C. For western blotting a HRP-conjugated Veriblot for IP secondary antibody (ab131366) (1/10000) was used to confirm successful immunoprecipation.

    See Abreview

  • IP sample preparation: Histone H3 (mono methyl K9) was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H3 (mono methyl K9) 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;

    Western blot conditions: 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab9045.

    Secondary antibody: IgG VeriBlot for IP secondary antibody (HRP) (ab131366) at 1/1000 dilution.

Protocols

References for VeriBlot for IP secondary antibody (HRP) (ab131366)

This product has been referenced in:
  • Kurtz CL  et al. microRNA-29 fine-tunes the expression of key FOXA2-activated lipid metabolism genes and is dysregulated in animal models of insulin resistance and diabetes. Diabetes N/A:N/A (2014). Read more (PubMed: 24722248) »
  • Jacky BP  et al. Identification of fibroblast growth factor receptor 3 (FGFR3) as a protein receptor for botulinum neurotoxin serotype A (BoNT/A). PLoS Pathog 9:e1003369 (2013). WB . Read more (PubMed: 23696738) »

See all 2 Publications for this product

Product Wall



Its likely you can used ab131366 to detect your primary mouse antibody. Just please note that ab131366 can react with the following isotypes, which does not include mouse IgG1:




Species Monoclonal Isotype(s)
Bovine...

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Application Immunoprecipitation
Myc tagged protein probed with a Myc polyclonal antibody and a secondary Rabbit IP specific antibody after Immunoprecipitation.
Lane 2 has a smaller Myc tagged protein and Lane 4 is IP control with no Myc tagged protein. The antibody recognizes the right protein with very little background (see image). I highly recommend this antibody.
Username

Abcam user community

Verified customer

Submitted Feb 28 2014

Abreviews
Application Immunoprecipitation
An IP was performed using ab175200, the sample was denatured in SDS sample buffer and boiled for 5 min. The sample was then run under denaturing conditions on a 10% gel. AB131366 was used as secondary antibody: 25ul in 5ml 5%-milk-TBST for 30 min at room temperature. No light chain was detected (~25kDa), a bit of heaychain (~50kDa) is still picked up by this antibody.
Username

Abcam user community

Verified customer

Submitted Nov 13 2013

Abreviews
Application Western blot
IRAK2 was immunoprecipitated from HEK293 cells (1 mg), run on an 8% gel and western blotted with the same antibody. The rabbit secondary was used at a dilution of 1/10000 before detection by the ECL method.
Username

Abcam user community

Verified customer

Submitted Nov 06 2013

Application Western blot
We performed an IP against Bim from HCT116 cell lysate. As the protein size of Bim is around 25 kDa, we first tested the VeriBlot secondary antibody (ab131366) to analyse its binding (lane 1). No signal from the IgG heavy and light chain, even after long exposure times, was observed when using ab131366. Next we developed the same membrane with a standard secondary antibody (ab97051) as a comparison (lane 2). The signal was much stronger with the standard secondary antibody but the strong signals of heavy and light chain of IgG overlayed the signal of Bim. To sum up, the VeriBlot secondary antibody is a nice tool to analyse proteins at around 25 or 55 kDa in IPs.

Lane 1 shows Bim long and short forms, after a exposure time of 5 min with ECL+. The secondary antibody (ab131366) was diluted 1:2000. Lane 2 shows Bim overlayed by the light chain of IgG after a exposure time of 2 min with ECL. The secondary antibody (ab97051) was diluted 1:10000.
Username

Mr. Christian Marx

Verified customer

Submitted Aug 27 2013

Thank you for your reply. Our Veriblot range really is the only line that we have that have been tested to provide the results you're looking for, so I will just issue you a credit for this purchase.


I am sorry that this antibody did no...

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Thank you for your reply and confirming these details.


I am happy to offer a replacement or credit. Please let me know which you would prefer.


I look forward to your reply so that I may assist you further.

Thank you for completing the questionnaire sent by my colleague. She has transferred your inquiry to me as I have additional experience in IP.


In reviewing the information you provided, I do agree that the secondary is detecting something...

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Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am at...

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Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.
In the event that a product is not functioning in the tested applications and species cited on the product d...

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