VeriBlot for IP Detection试剂(HRP) (ab131366)

概述

  • 产品名称
    VeriBlot for IP Detection试剂(HRP)
  • 偶联物
    HRP
  • 经测试应用
    适用于: WBmore details
  • 常规说明

    VeriBlot for IP Detection Reagents 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 detection reagents only recognize native (non-reduced) antibodies and therefore the detection of heavy and light chains is highly minimized, if the immunoprecipitate is fully reduced.

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

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

    Species Monoclonal Isotype(s)
    Bovine IgG2
    Goat IgG2
    Human IgG1, IgG2, IgG4
    Mouse

    IgG2a, IgG2b, IgG3  (IgG1 affinity may or may not be strong

    so it should be empirically tested)  

    Rat IgG2C
    Rabbit Total IgG
    Sheep IgG2


    2. The VeriBlot for IP Detection Reagent (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. Boil samples for 5-10 minutes in SDS sample buffer with a increase in SDS amount if required.


    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

性能

应用

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.

应用 Ab评论 说明
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.

Make sure the lysates are reduced and denatured completely.  

图片

  • 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 Detection Reagent (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 Detection Reagent (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 Detection Reagent (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.

    Detection: VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution.

实验方案

文献

This product has been referenced in:
  • Dimartino D  et al. The Long Non-coding RNA lnc-31 Interacts with Rock1 mRNA and Mediates Its YB-1-Dependent Translation. Cell Rep 23:733-740 (2018). Read more (PubMed: 29669280) »
  • Mir R  et al. The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human. Biochem J 475:1861-1883 (2018). Read more (PubMed: 29743203) »
See all 21 Publications for this product

客户评价及客户问答

1-10 of 13 Abreviews or Q&A

Abreviews
Application
Western blot
Works very well.
The IP was done with Rabbit antibody and afterwards WB was also done with rabbit antibody. can't see the light and heavy chains of the IPed antibody.

Abcam user community

Verified customer

提交于 Oct 04 2016

Answer

A1: To eliminate this the samples should be denatured and reduced completely. The Veriblot antibodies does not bind reduced IgGs so please make sure the samples are boiled for 5-10 minutes at 100C and the SDS is of high quality. If required amount of SDS can be increased in the samples buffer.

A2: Nonspecific bands are typically due to excess HRP in the system, or excess of primary antibody. SDS may also cause proteins to bind non-specifically as surfaces that normally would not be exposed are opened up with the linearization of the protein, which can result in protein: protein interactions that would not occur in the native state. Running a native or non-denaturing gel, should eliminate this problem with SDS.

Addition of 0.05% non-ionic detergent such as Tween 20 or Triton X-100 in the blocking and washing solution is highly recommended.

Proper multiple washings of membrane are recommended.

A3: The affinity with mouse IgG1 isotype is low as compared to other listed Isotype so we are unable to guarantee it. It may work, but has to be tested empirically on a case by case basis.

Read More
Application
Immunoprecipitation
Mitochondria were isolated from HEK293 cells and 200µg extract was used per IP. 1µg ATP synthase beta (ab14730) was crosslinked to a mixture of Protein A and G beads (left panel on figure) or not crosslinked (right panel on figure). GAL4 was used as a negative control. The extract was incubated with the beads and antibodies overnight at 4C in 100mM Tris, pH 7.6, 20% glycerol and inhibitors. In the morning the beads were washed 3 times and eluted with 1M glycine, pH 2. The samples were run on 12% gels at 180V for 1h, semidry transferred on a nitrocellulose membrane for 30min at 25V and blocked in 5% milk for 30min at room temperature. The membranes were incubated with 1:1000 dilution of ATP synthase alpha (ab14748) and ATP synthase beta (ab14730) antibodies in BSA overnight at 4C. In the morning the membranes were washed 3 times for 10 min in TBST and incubated with secondary antibody for 1h in 5% milk for the membrane with the crosslinked beads or with Veriblot (ab131366) for the non-crosslinked samples. After another 3 washes for 10min in TBST the signal was detected using ECL detection reagent.

Abcam user community

Verified customer

提交于 Oct 28 2014

Answer



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 IgG2
Goat IgG2
Human IgG1, IgG2, IgG4
Mouse IgG2a, IgG2b, IgG3
Rat IgG2C
Rabbit Total IgG
Sheep IgG2



If your mouse primary antibody is IgG1, then you can detect it with ab131368.

I hope this helps. Please let me know if you have any further questions.

Read More
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.

Abcam user community

Verified customer

提交于 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.

Abcam user community

Verified customer

提交于 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.

Abcam user community

Verified customer

提交于 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.

Mr. Christian Marx

Verified customer

提交于 Aug 27 2013

Answer

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 not perform as stated on the datasheet, I have asked our Finance department to issue a credit note for you. The credit note may be used in one of the following ways:

(1) Redeemed against the original invoice if this hasn't already been paid.
(2) Held on the account for use against a future order.
(3) A full refund can be offered where no other invoices are outstanding.

Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge.

To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website.

The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

Read More

Answer

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.

Read More

1-10 of 13 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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