Overview

  • Product name
  • Description
    Rabbit polyclonal to Citrulline
  • Host species
    Rabbit
  • Specificity
    Using conjugated citrulline glutaraldehyde Protein, antibody specificity was performed with an ELISA test by competition experiments with the following compounds:
    CompoundCross-reactivity ratio
    Citrulline-G-BSA1
    Arginine-G-BSA, Glutamine-G-BSA, Ornithine-G-BSA1 > 100,000
    Conjuaged Citrulline and conjugated Homo-citrulline1:1000
    G = Glutaraldehyde, BSA = Bovine Serum Albumin
  • Tested applications
    Suitable for: ICC/IF, IHC-P, ELISA, IHC-Frmore details
  • Immunogen

    Chemical/ Small Molecule by a Glutaraldehyde linker.

  • General notes
    This polyclonal antibody recognizes only citrulline residues on proteins not free citrulline.

Properties

Applications

Our Abpromise guarantee covers the use of ab6464 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
ICC/IF Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
ELISA 1/5000 - 1/10000.
IHC-Fr 1/1000 - 1/5000.

Target

  • Relevance
    The amino acid Citrulline is required to detoxify the liver from ammonia, which is a waste product of the body from oxidation. Citrulline promotes energy and assists with the immune system. This unusual amino acid is formed in the urea cycle by the addition of carbon dioxide and ammonia to ornithine. It is then combined with aspartic acid to form arginosuccinic acid, which later is metabolized into the amino acid arginine.

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of breast DCIS tumor with apoptotic center labeling Citrulline  with ab6464 at 1/100 dilution. Tissue was fixed in 10% buffered formalin for 24 hrs then transferred to 70% ethanol until processed and embedded and permeabilized with Tris Buffered Saline with 0.025% Triton X-100. Heat mediated antigen retrieval was performed using 10mM Sodium Citrate Buffer with 0.05% Tween 20 pH 6.0. Tissue was blocked with TBS with 10% normal (secondary species) serum and 1% BSA for 1 hour at 37°C, followed by incubation with Anti-Citrulline antibody (ab6464) in TBS with 1% BSA for 18 hours at 4°C at 1/100 dilution. A polyclonal goat anti rabbit IgG biotin conjugate secondary antibody was used at 1/200 dilution. 

     

    See Abreview

  • Immunofluorescence analysis of THP-1 cells, staining Citrulline with ab6464.

    Cells were fixed with paraformaldehyde and blocked with 0.05% BSA for 1 hour at 37°C. Samples were incubated with primary antibody (1/500 in diluent) for 2 hours at 37°C. ab6717 Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (FITC) was used to detect staining.

    See Abreview

References

This product has been referenced in:
  • Mohamed BM  et al. Induction of protein citrullination and auto-antibodies production in murine exposed to nickel nanomaterials. Sci Rep 8:679 (2018). Read more (PubMed: 29330439) »
  • Verheul MK  et al. Pitfalls in the detection of citrullination and carbamylation. Autoimmun Rev 17:136-141 (2018). Read more (PubMed: 29203292) »
See all 10 Publications for this product

Customer reviews and Q&As

1-10 of 24 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Vein)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate based (Vector Laboratories H-3300)
Permeabilization
Yes - 0.2% Triton-X 10 mins RT
Specification
Vein
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jan 29 2018

Application
Western blot
Sample
Human Recombinant protein (Histone H3)
Gel Running Conditions
Non-reduced Denaturing
Loading amount
1 µg
Treatment
1µg PAD4 for 24 hrs
Specification
Histone H3
Blocking step
Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Mona Biermann

Verified customer

Submitted Sep 26 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 37°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10mM Sodium Citrate Buffer with 0.05% Tween 20 pH 6.0
Sample
Human Tissue sections (Breast, DCIS tumor with apoptotic center)
Specification
Breast, DCIS tumor with apoptotic center
Permeabilization
Yes - Tris Buffered Saline with 0.025% Triton X-100
Fixative
Formaldehyde

Ms. Lynne Anguish

Verified customer

Submitted Jun 09 2014

Answer

Thank you for contacting us. We currently have two antibodies that will detect the citrulline modification on any proteins: ab6464 and ab100932. While ab6464 has been validated in IHC on frozen tissue sections, we have not tested this antibody for use in WB. Similarly, ab100932 has been validated in WB but not in IHC. If you would like to test either of these antibodies, I am happy to offer you our 100% testing discount program. If you purchase the antibody for full price and submit an Abreview with your protocol and results using the antibody in an untested application, you can get a discount code for a free primary antibody on a future purchase. Whether or not the antibody works, the next antibody you buy from us would be free. If you are interested in this program, please reply and let me know which antibody you would like to test, and I will be happy to set up the code for you.

Read More

Answer

Thank you for your reply and this complement of information.

ab6464 has not been tested in Western Blot and may not be suitable for this application. This would explain why you don't see any staining with this antibody.
The information on the product datasheet as well as the encouraging positive results obtained in Western Blot suggest that ab100932 is indeed capable of targeting the citrulline independently of the amino acid sequence.

An optimisation of the incubation time and conditions for the antibody ab100932 in ELISA may be necessary in this case.

Please let me know about the new trials with the antibodies in ELISA. If the problem stays the same I will be happy to issue a credit note against the original order.

Read More

Answer

Thank you for contacting us.

The source of the antibody informed us that they have never tested the antibody by coating plates with fibrinogen.
Their testing are made using BSA and citrunilated BSA. However, the testing conditions are very similar to yours. The difference is that the lab do not perform a washing step before the saturation with BSA.
I attached to this email the results obtained during the validation of batch GR22466 of this antibody ab6464 as well as an ELISA protocol provided by the lab.

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

Read More

Answer

Thank you for your email.

I am very happy to hear that the protocol optimizations improved the results and the antibody is now working fine.

I wish all the best for future experiments and research.

Read More

Answer

Thank you for your inquiry! We are well, thanks- Ihope you are well too!

The two products are from two different laboratories and therefore productions. They are both against bound citrulline and guaranteed by us in ICC/IF, ELISA, IHC-Fr for the ab6464 and ELISA, ICC, WB for the ab100932. If the customer wants to do Western blot, I can recommend the ab100932. If the customer wants to do frozen section immunohistochemistry, I recommend the ab6464.

Other differences are the information or the unit size. The ab100932 comes in 200ul and for the ab6464 we have the crossreactivity data against homocitrulline for example on the datasheet.

I hope this information is helpful. Please do not hesitate to contact us again should you have any other question.

Read More

Question

I am also sorry about that because I have good experience with your antibodies. It was surprising for me as well that even the new antibody can not recognize the antigen ... I thought that there might be some problem in our protocol, but I can not find it. I filled out the questionery and I hope that we can figure out together what is going on ... It would be very urgent as some clinical samples are waiting for us and the deadline for the measurements is the end of June.
1. protocol
1) Abcam product code: ab6464
2) Abcam order reference number or product batch number: 1092015
3) Description of the problem: we can not get positive signal using the antibody
4) Type of ELISA: indirect
5) Sample details: citrullinated-peptides and citrullinated BSA
6) Target antigen (if found in heterogeneous sample): citrullinated proteins
7) Positive control: in house citrullinated peptides with PAD4 enzyme
8) Negative control: PBS coating (no antigen)
9) Blocking agent (eg BSA…):
Concentration: 1% milk powder in TBS
Blocking time: 1 hour
Blocking temperature: room temperature
10) Capture or Primary antibody information
Used to coat wells? No
Species: polyclonal rabbit IgG
Reacts against: citrulline
Concentration or dilution: 1/5000
Diluent buffer: 0.1% milk-TTBS
Incubation time: 1 hour
Incubation temperature: room temperature
11) Detection antibody:
Species:
Reacts against:
Concentration or dilution:
Diluent buffer:
Incubation time:
Incubation temperature:
Conjugation:
12) Enzyme labeled secondary antibody:
Species: sheep polyclonal secondary IgG H&L
Reacts against: rabbit IgG
Concentration or dilution : 1/5000
Diluent buffer : 0.1% milk-TTBS
Incubation time: 1 hour
Incubation temperature: room temperature
Conjugation: alkaline phosphatase
13) Washing after primary and secondary antibodies:
Buffer: TTBS
Number of washes: 3
Length of washes: 10 seconds
14) Substrate: 1 mM pNPP in 30 mM NaOH
Incubation time: 30 minutes in dark at room temperature
15) How many times have you run this ELISA? 6 times
Do you obtain the same results every time? We always get negative results.
What steps have you altered to try and optimize the use of this antibody? Different dilutions of the antibody, overnight coating, increasing the incubation time.
2. protocol
1) Abcam product code: ab6464
2) Abcam order reference number or product batch number: 1092015
3) Description of the problem: we can not get positive signal using the antibody
4) Type of ELISA: indirect
5) Sample details: CCP (CCPlus® Immunoscan from EuroDiagnostica)
6) Target antigen (if found in heterogeneous sample): CCP
7) Positive control:
8) Negative control:
9) Blocking agent (eg BSA…): no blocking was required
Concentration:
Blocking time:
Blocking temperature:
10) Capture or Primary antibody information
Used to coat wells? No
Species: polyclonal rabbit IgG
Reacts against: citrulline
Concentration or dilution: 1/5000
Diluent buffer: 0.1% milk-TTBS
Incubation time: 1 hour
Incubation temperature: room temperature
11) Detection antibody:
Species:
Reacts against:
Concentration or dilution:
Diluent buffer:
Incubation time:
Incubation temperature:
Conjugation:
12) Enzyme labeled secondary antibody:
Species: sheep polyclonal secondary IgG H&L
Reacts against: rabbit IgG
Concentration or dilution : 1/5000
Diluent buffer : 0.1% milk-TTBS
Incubation time: 1 hour
Incubation temperature: room temperature
Conjugation: alkaline phosphatase
13) Washing after primary and secondary antibodies:
Buffer: TTBS
Number of washes: 3
Length of washes: 10 seconds
14) Substrate: 1 mM pNPP in 30 mM NaOH
Incubation time: 30 minutes in dark at room temperature
15) How many times have you run this ELISA? Twice.
Do you obtain the same results every time? Yes.
What steps have you altered to try and optimize the use of this antibody? None
3. protocol
1) Abcam product code: ab6464
2) Abcam order reference number or product batch number: 1092015
3) Description of the problem: we can not get positive signal using the antibody
4) Type of ELISA: indirect
5) Sample details: CCP (CCPlus® Immunoscan from EuroDiagnostica)
6) Target antigen (if found in heterogeneous sample): CCP
7) Positive control:
8) Negative control:
9) Blocking agent (eg BSA…): no blocking was required
Concentration:
Blocking time:
Blocking temperature:
10) Capture or Primary antibody information
Used to coat wells? No
Species: polyclonal rabbit IgG
Reacts against: citrulline
Concentration or dilution: 1/5000
Diluent buffer: Sample diluent provided with the kit
Incubation time: 1 hour
Incubation temperature: room temperature
11) Detection antibody:
Species:
Reacts against:
Concentration or dilution:
Diluent buffer:
Incubation time:
Incubation temperature:
Conjugation:
12) Enzyme labeled secondary antibody:
Species: goat polyclonal antibody
Reacts against: rabbit IgG
Concentration or dilution : 1/2000
Diluent buffer : Sample diluent provided with the kit
Incubation time: 30 minutes
Incubation temperature: room temperature
Conjugation: HRP
13) Washing after primary and secondary antibodies:
Buffer: TTBS
Number of washes: 3
Length of washes: 10 seconds
14) Substrate: 100 ul TMB
Incubation time: 30 minutes in dark at room temperature
Stop solution: 50 ul 1 mM H2SO4
15) How many times have you run this ELISA? Twice.
Do you obtain the same results every time? Yes.
What steps have you altered to try and optimize the use of this antibody? We applied 1/5000 and 1/10000 primary antibody dilutions.
In case of CCP (protocols 2 and 3) no positive control was used. From this batch the immunological laboratory of the University routinely uses the wells for anti-citrulline antibody detection from the serum of RA patients and it works well.
All the absorbances recorded in all protocols were less than 0.1
Thank you for your help!
Best regards,

Read More
Answer

Thank you very much for filling in the questionnaire.

The lab is using the belowprotocol to test the antibody. I noticed there are differences, particular concerning the buffers. The positive control is Citrulline-BSA.

Maybe this protocol will help improve the results. Please let me know how you get on.

ELISA protocol:

1 - Coating of Immunogen(BSA) (10µg/ml) in maxisorp well plates with a solution of sodium carbonate buffer 0.05M (pH 9,6), during sixteen hours at 4°C.

2 -Saturation of well plates with of a solution of PBS (pH 7,35) containing 2.5g/l of BSA , 10% and 0.5% of Tween (one hour at 37°C).

3 - Wash with PBS containing 0.5% of Tween (PBS Tween) (three times).

4 -Antibody will be diluted (1/2,000 – 1/10,000) in PBS Tween containing 2.5g/l BSA, 200µl by well plate (incubating during 2 hours at 37°C).

5 - Wash with PBS Tween (three times).

6 - 200µl of peroxidase-labelled goat anti-rabbit diluted (1/10,000) in a solution of PBS Tween containing 1g/l of BSA, will be applied by well plate (during one hour at 37°C)

7 - Well plates will be rinsed with PBS Tween (three times).

8 - And finally the peroxidase will be developed by incubating 200µl by well plate of a citrate 0,1M/phosphate 0,2M (pH 5) solution containing 0.4% of OPD and 0.03% of hydrogen peroxide for ten minutes in the dark, after that, we will stop the reaction by the addition of 50µl of 2M HCl.

9 - The optical density will be measured at 492nm.

Competitive ELISA protocol:

The same protocol is used, but modifications occur in stage “4”. The day before the test, competition scales are made with each competitor (10-1 - 10-10g/l of BSA conjuguate) and diluted antibody. These tubes are incubated at 4°C overnight. The results are expressed as OD with competitor/ OD without competitor for each point of competition scale.

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Skin)
Specification
Skin
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: EDTA pH 9.0
Permeabilization
No
Blocking step
Casein as blocking agent for 15 minute(s) · Concentration: 0.25% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jun 15 2012

1-10 of 24 Abreviews or Q&A

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