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Immunology Immunoglobulins Heavy Chain IgG

VeriBlot for IP Detection试剂(HRP) (ab131366)

  • Datasheet
  • SDS
Reviews (7)Q&A (5)References (81)

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Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)

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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  

    Note: If using mouse IgG1, perform a dot blot to determine compatibility. VeriBlot for IP Detection Reagent (HRP) might not detect mouse IgG1.  

    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.

    Note: If denatured and blotted IgG are not clearly detected, the following steps may be used to increase the amount of denatured IgG in the sample:

    - Increase the concentration of reducing agent

    - Boil sample to aid in reduction of IgG disulfide bonds

    - Use dentaturing electrophoresis conditions

    A full troubleshooting guide is available here.

性能

  • 形式

    Liquid
  • 存放说明

    Shipped at 4°C. Store at +4°C.
  • 存储溶液

    Constituent: 1% MOPS
  • Concentration information loading...
  • 研究领域

    • Immunology
    • Immunoglobulins
    • Heavy Chain
    • IgG
    • Secondary antibodies
    • anti-Rabbit
    • IgG
    • Enzyme
    • HRP
    • Isotype/Loading Controls
    • Isotype Controls
    • Rabbit

相关产品

  • Related Products

    • Donkey Anti-Rabbit IgG H&L (HRP) (ab16284)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab131366于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
WB (3)
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.  

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

  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
    Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)This image is courtesy of an anonymous Abreview.

    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

  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
    Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)This image is courtesy of an Abreview submitted by Christian Marx.

    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

  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
    Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)This image is courtesy of an anonymous 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

  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
    Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)

    ab124962 (purified) at 1/20 immunoprecipitating IL-1RA in NIH/3T3 whole cell lysate.
    Lane 1 (input): NIH/3T3 whole cell lysate (10µg)
    Lane 2 (+): ab124962 + NIH/3T3 whole cell lysate.
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab124962 in NIH/3T3 whole cell lysate.
    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.
    Blocking buffer and concentration: 5% NFDM/TBST.
    Diluting buffer and concentration: 5% NFDM /TBST.

  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
    Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)

    ab108338 (purified) at 1/20 dilution (2µg) immunoprecipitating ATG9A in HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate.
    Lane 1 (input): HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10µg
    Lane 2 (+): ab108338 & HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab108338 in HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate
    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution. No band in input lane is due to the boiled lysates
    Blocking and diluting buffer: 5% NFDM/TBST.

  • Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)
    Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (ab131366)

    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

  • Immunoprecipitation protocols
  • Western blot protocols

Click here to view the general protocols

数据表及文件

  • SDS download

  • Datasheet download

    Download

文献 (81)

发表研究结果有使用 ab131366?请让我们知道,以便我们可以引用本数据表中的参考文章。

ab131366 被引用在 81 文献中.

  • Jansens RJJ  et al. Alphaherpesvirus US3 protein-mediated inhibition of the m6A mRNA methyltransferase complex. Cell Rep 40:111107 (2022). WB, IP, ICC ; Pig . PubMed: 35858564
  • Jacomin AC  et al. Degradation of arouser by endosomal microautophagy is essential for adaptation to starvation in Drosophila. Life Sci Alliance 4:N/A (2021). PubMed: 33318080
  • Tan CT  et al. MOAP-1-mediated dissociation of p62/SQSTM1 bodies releases Keap1 and suppresses Nrf2 signaling. EMBO Rep 22:e50854 (2021). PubMed: 33393215
  • Mohapatra P  et al. CMTM6 drives cisplatin resistance by regulating Wnt signaling through the ENO-1/AKT/GSK3ß axis. JCI Insight 6:N/A (2021). PubMed: 33434185
  • Campbell AE  et al. Temporal modulation of the NF-?B RelA network in response to different types of DNA damage. Biochem J 478:533-551 (2021). PubMed: 33438746
View all Publications for this product

客户评价及客户问答

Show All 评价 Q&A
提交评价 提交问题

1-10 of 12 Abreviews or Q&A

Western blot using Veriblot secondary

Good Excellent 5/5 (Ease of Use)
Abreviews
Abreviews
abreview image
Immunoprecipitations were performed and samples were incubated at 95°C for 5 minutes in sample buffer containing SDS and beta-mercaptoethanol to fully reduce proteins. Samples were run on an SDS-PAGE gel and Western blots were performed, using Veriblot as the secondary antibody.
There were two IP samples.
Lane 1 - protein target MW: 75 kDa
Lane 2 - protein target MW: 22 kDa.
For sample 2, which IP'ed very well, the heavy chain signal was very low. For sample 1, however, which did not IP well, the heavy chain signal was very high. The asterisks mark the IP'ed protein. In both cases the light chain signal was very low or non-existent.
For proteins that do not IP well, are low abundance, or have poor primary antibodies, expect to see the heavy chain band. In those cases, as long as your protein of interest is not at ~50 kDa this product will work. If your protein is ~50 kDa, I would recommend using a secondary antibody that only recognizes the light chain (~25 kDa) instead.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Feb 12 2021

veriblot with rabbit antibody

Excellent
Abreviews
Abreviews
abreview image
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Oct 04 2016

Comparing IP signal with crosslinked antibodies to beads or using Veriblot to avoid detection of IgG heavy and light chain

Good
Abreviews
Abreviews
abreview image
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Oct 28 2014

Western blot using the antibody with cell lysate prepared from S. cerevisiae.

Excellent
Abreviews
Abreviews
abreview image
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Feb 28 2014

AB131366 as secondary antibody for IP

Excellent
Abreviews
Abreviews
abreview image
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Nov 13 2013

WB of IRAK2 IP

Excellent
Abreviews
Abreviews
abreview image
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Nov 06 2013

Detection of immunoprecipitated Bim with Anti-rabbit IgG VeriBlot secondary antibody

Excellent
Abreviews
Abreviews
abreview image
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

MR. Christian Marx

Verified customer

提交于 Aug 27 2013

Question

Q1: Why I see band of approximately same size as of heavy chain?
Q2: Why there are non-specific bands on the gel?
Q3: Why mouse IgG1 is not listed in specificty table?

Read More

Abcam community

Verified customer

Asked on Dec 30 2014

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

Padamjeet Singh

Abcam Scientific Support

回复于 Dec 30 2014

Question

I want an anti mouse HRP antibody which will not detect Heavy and Light chain
is https://www.abcam.com/veriblot-for-ip-secondary-antibody-hrp-ab131366.html the right one is it an antimouse? or do you have 2nd antibodies HRP that I can use to avoid detecting Heavy or light chain or both.

Read More

Abcam community

Verified customer

Asked on Apr 23 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

Jeremy Kasanov

Abcam Scientific Support

回复于 Apr 23 2014

Question

If you have a secondary antibody against rabbit or goat that works (is not detecting denatured IgG from the IP pulldown) then we would be glad to have that. We are facing huge problems detecting 25 kd proteins from IP samples. If not we would like the credit.

Read More

Abcam community

Verified customer

Asked on Aug 02 2012

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

Abcam Scientific Support

回复于 Aug 02 2012

1-10 of 12 Abreviews or Q&A

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