Overview

  • Product name

  • Description

    Rabbit polyclonal to C4d
  • Host species

    Rabbit
  • Specificity

    Recognizes C4d-A and C4d-B.
  • Tested applications

    Suitable for: IHC-P, Flow Cyt, ICC/IF, IHC-Frmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to Human C4d aa 1223-1237 (C terminal) conjugated to keyhole limpet haemocyanin. Region 100% conserved in C4d-B (Uniprot: P0C0L5)
    Sequence:

    ETGDNLYWGSVTGSQ


    Database link: P0C0L4

  • Positive control

    • Glomerular mesangial tissue

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer

    pH: 7.20
    Preservatives: 0.09% Sodium azide, 0.1% Proclin
    Constituent: PBS
  • Concentration information loading...
  • Purity

    Protein G purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab36075 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
IHC-P 1/20 - 1/80. Antigen retrieval with a citrate buffer system (or equivalent) is recommended.
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 1/20 - 1/80.
IHC-Fr 1/20 - 1/80.

Target

  • Function

    C4 plays a central role in the activation of the classical pathway of the complement system. It is processed by activated C1 which removes from the alpha chain the C4a anaphylatoxin. The remaining alpha chain fragment C4b is the major activation product and is an essential subunit of the C3 convertase (C4b2a) and the C5 convertase (C3bC4b2a) enzymes of the classical complement pathway.
    Derived from proteolytic degradation of complement C4, C4a anaphylatoxin is a mediator of local inflammatory process. It induces the contraction of smooth muscle, increases vascular permeability and causes histamine release from mast cells and basophilic leukocytes.
  • Involvement in disease

    Defects in C4A are the cause of complement component 4A deficiency (C4AD) [MIM:120810]. A rare defect of the complement classical pathway associated with the development of autoimmune disorders, mainly systemic lupus with or without associated glomerulonephritis.
  • Sequence similarities

    Contains 1 anaphylatoxin-like domain.
    Contains 1 NTR domain.
  • Post-translational
    modifications

    Prior to secretion, the single-chain precursor is enzymatically cleaved to yield the non-identical chains (alpha, beta and gamma). During activation, the alpha chain is cleaved by C1 into C4a and C4b, and C4b stays linked to the beta and gamma chains. Further degradation of C4b by C1 into the inactive fragments C4c and C4d blocks the generation of C3 convertase.
    N- and O-glycosylated. O-glycosylated with a core 1 or possibly core 8 glycan.
  • Cellular localization

    Secreted.
  • Information by UniProt
  • Database links

  • Alternative names

    • acidic C4 antibody
    • Acidic complement C4 antibody
    • basic C4 antibody
    • basic complement C4 antibody
    • C3 and PZP-like alpha-2-macroglobulin domain-containing protein 2 antibody
    • C3 and PZP-like alpha-2-macroglobulin domain-containing protein 3 antibody
    • C4 antibody
    • C4, chido form antibody
    • C4, Rodgers form antibody
    • C4-1 antibody
    • C4a anaphylatoxin antibody
    • C4A antibody
    • C4A2 antibody
    • C4A3 antibody
    • C4A4 antibody
    • C4A6 antibody
    • C4AD antibody
    • C4b antibody
    • C4B_2 antibody
    • C4B1 antibody
    • C4B12 antibody
    • C4B2 antibody
    • C4B3 antibody
    • C4B5 antibody
    • C4BD antibody
    • C4d antibody
    • C4d fragment antibody
    • C4F antibody
    • C4S antibody
    • CH antibody
    • Chido form of C4 antibody
    • CO4 antibody
    • CO4A_HUMAN antibody
    • Complement C4 A antibody
    • Complement C4 B antibody
    • Complement C4 gamma chain antibody
    • complement C4-A antibody
    • complement C4-B antibody
    • complement C4-B-like antibody
    • complement C4B1a antibody
    • Complement component 4A antibody
    • complement component 4A (Rodgers blood group) antibody
    • Complement component 4B antibody
    • complement component 4B (Chido blood group) antibody
    • complement component 4B (Chido blood group), copy 2 antibody
    • complement component 4B (Chido blood group)-like antibody
    • Complement component 4F antibody
    • Complement component 4S antibody
    • CPAMD2 antibody
    • CPAMD3 antibody
    • DADB-112B14.11 antibody
    • MGC164979 antibody
    • RG antibody
    • Rodgers form of C4 antibody
    see all

Images

  • Immunohostochemical analysis of C4d expression in paraffin embedded formalin fixed human glomerular mesangial tissue, using 1/40 ab36075. Staining developed using high sensitivity Avidin/Biotin detection system.
  • ab36075 staining C4d in human kidney cells by Immunocytochemistry/ Immunofluorescence (snap frozen). The cells were acetone fixed. Samples were incubated with primary antibody at 1/60 for 1 hour at 25°C. The secondary antibody used was an IgG conjugated to Alexa Fluor® 488.
  • Ab36075 staining C4d in Human kidney tissue sections by Immunohistochemistry (IHC-P- paraformaldehyde-fixed, paraffin embedded sections). Tissue was fixed with formaldehyde and heat mediated antigen retrieval was performed at 100°C with citrate for 20 minutes. Primary antibody was incubated at 1/80 dilution for 20 minutes at 25°C. A HRP conjugated anti-mouse IgG was used as a secondary antibody.

    See Abreview

  • Ab36075 staining C4d in Human nasal sinus tissue by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with acetone and blocked with 20% milk for 20 minutes. Primary antibody was incubated at a 1/80 dilution in PBS for 1 hour at 25°C. A biotin conjugated goat anti-rabbit was used as a secondary antibody at 1/100 dilution.

    See Abreview

References

This product has been referenced in:

  • Zhu X  et al. All-trans retinoic acid protects mesenchymal stem cells from immune thrombocytopenia by regulating the complement-IL-1ß loop. Haematologica N/A:N/A (2019). Read more (PubMed: 30679324) »
  • Jiang Y  et al. The Effect of Histological CD20-Positive B Cell Infiltration in Acute Cellular Rejection on Kidney Transplant Allograft Survival. J Immunol Res 2016:7473239 (2016). Read more (PubMed: 28058267) »
See all 4 Publications for this product

Customer reviews and Q&As

1-10 of 20 Abreviews or Q&A

Answer

Thank you for the reply.

Could you please confirm whether either of the vials of ab36075 purchased showed any staining (one of the 20xxxx and the one from the xxxxx)?

Also, I asked for some feedback of how the antibodies had been stored. Has the customer provided this information?

I look forward to receiving your reply.

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Answer

Thank you for contacting us. The antibody is supplied in liquid form and does not have to be reconstituted. The vial should contain 50 ul (0.2 mg/ml), if you do not see it you should spin it down. I can direct you to a starting dilution if you can tell me what application you plan to use.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for your reply.

Having reviewed this case it seems that using the protocol the customer has applied the antibody should be producing staining.

May I ask, how has the antibody been stored? At what temperature and was it aliquotted?

The order details you gave to xxxx refers to an order of the 24th of September 2010. Was the antibody used before the initial complaint was made in April of this year? What were the results of these experiments?

Many thanks for your cooperation, I look forward to receiving your reply.

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Question
Answer

Thank you very much for having followed up with the customer and provided this additional information.

When looking through the protocol, I was surprised about the information on the secondary antibody. The customer mentioned to have used a rabbit anti human secondary antibody. This would not give a staining, as the primary antibody is a rabbit antibody. For a secondary antibody to detect the ab36075 an anti rabbit (e.g. goat anti rabbit, or donkey anti rabbit, ..) must be used. Can you please confirm this?

If the customer obtains no signal with an anti rabbit secondary antibody, we will be pleased to replace or credit this antibody for them. I apologize for the inconvenience.

For future experiments, I can also recommend strongly to the customer to include a blocking step. Indeed, blocking is a very crucial step for obtaining good images. Most of the time, 10% serum block is appropriate. As serum, we recommend to use serum of the species in which the secondary antibody is made (if a goat anti rabbit is used, we recommend therefore to use a goat serum to block). For further details or tips, please recommend the customer also our online protocols. https://www.abcam.com/index.html?pageconfig=resource&rid=11384

Thank you very much for your cooperation. I look forward to hear back from you.

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Question
Answer

We have previously been in contact regarding ab36075, which was not providing satisfactory results.

A free of charge replacement vial was issued and I am writing to inquire if the replacement product has worked. I would appreciate if you could let me know whether satisfactory results were obtained with this new vial as I am keen to know how you are progressing.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, please do not hesitate to let us know.

I wish you the best of luck with your research and please do not hesitate to contact us if you have any further questions.

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Question
Answer

Thank you for contacting us.

Your credit note ID is XXXXXXX.

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

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Answer

Thank you for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. The only thing I can suggest now is manual staining with Microwave antigen retrieval method.

I apologize for the inconvenience and am pleased to offer you a credit note, or refund in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Answer

Thank you for this additional information.

I would be pleased to investigate this issue further to see whether we could provide some protocol tips. Can you please therefore send us the protocol details with help of the filled questionnaire (it should not take more than 5 minutes to fill) as well as an image of the result? This will help us to see whether there are any modifications which could significantly improve the results. If we are unable to improve the results, we will replace the antibody for the customer. Can you actually double check the order number you provided? I found there was a more recent order last year as well.

Thank you for your cooperation. I am looking forward to hear back from you.

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Question

Thank you for your quick response. Yes , we tried the staining as per recommended protocol and conditions/ dilutions provided by the abcam with both the antibodies with frozen mouse tissue.



The nature of the problem is that the staining with Twist Ab looks similar and non specific as it appears with the isotype control mouse antibody.Same is the case with the C4d ab that there looks no difference in staining with C4D Ab and with comparable isotype control.





Rehan Kahloon MD
Division of Pulmonary Allergy and Critical Care
Department of Medicine
University of Pittsburgh Medical Center
kahloonra@upmc.edu
412-716-9311


________________________________
From: technical@abcam.com [technical@abcam.com]
Sent: Thursday, April 19, 2012 5:12 PM
To: Kahloon, Rehan
Subject: Reply from Abcam to your enquiry regarding ab36075, ab50887 [CCE3700093]

[abcam: discover more]

Dear Rehan

Thank you for contacting Abcam.

Under our Abpromise we do guarantee our products to work as stated on the datasheet and if for whatever reason they do not work as stated and we cannot help resolve the issue, then we will be happy to either replace or refund the cost of the product. The Abpromise is good for 6 months.

To be able to better assist you in this matter, could you please provide me with information as to how you were using these antibodies, eg western blot, IHC-P, IP etc and also which species your sample came from. If you could also include a brief summary of the nature of the problems you are having, as well as the protocol that you have been using, that would be most helpful.

I look forward to your reply and helping you resolve these issues.

Help us improve our service.
Rate your experience with us today.

Best regards,
John

John Constable
Scientific Support Specialist
Abcam Inc.
www.abcam.com

Your original inquiry to Abcam:

Dear Customer Service,



I am writing in reference to our experience with two of the Antibodies we recently ordered from Abcam.



1- Anti- Twist Ab Catalog 50887 P.O. number 1139131.

2-C4D Antibody Ab Catalog Number ab 36075 P.O. Number 1135999.



Despite repeated attempts and following all the optimal protocols provided, we have not be able to make these antibodies work .



It would be great if we can get our credit back or have them returned, since we believe they are not effective.



Thanks







Rehan Kahloon MD
Division of Pulmonary Allergy and Critical Care
Department of Medicine
University of Pittsburgh Medical Center
WBST1200-23B
200 Lothrop Street.
Pittsburgh,PA.
kahloonra@upmc.edu
412-716-9311







Abcam Customer Services and Scientific Support Team
www.abcam.com/technical

[CCE3700093]




Discover more at abcam.com

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Answer

Thank you for your response.

I am sorry to hear that you have been experiencing problems when using our products. Our anti-Twist antibody in guaranteed to work in mouse tissues in IHC-Fr. We have added recommended dilutions or specific protocols for this producthowever there are customer’s reviews in our Abreview section which do give some protocol information that these customers have used. I noticed differences between these Abreview protocols and was wondering if you could provide me with the protocol steps which you have used, any troubleshooting step that you taken and any images should you have these. If we cannot solve the issues which you are experiencing, I will be glad to send you a free of charge replacement, credit note or refund under our Abpromise guarantee.

We currently do not recommend using ab36075 anti-C4d on mouse tissues. The homology for the immunogen used to create this product and the mouse protein is below the threshold for cross reactivity. As such, I am very happy to look over your protocol and provide tips and suggestions which may help. However this product would not be covered by our Abpromise.

You are doing mouse on mouse staining with these products.Staining of mouse tissue using mouse antibody is a complicated process as high levels of background are often observed. It is notoriously difficult to eliminate this background.I would strongly recommend these tips for eliminating background when staining mouse tissue with a mouse monoclonal.

-1. Block endogenous IgG withunconjugated Fab fragment Anti-Mouse IgG (H+L) for 1 hr at room temperature, or overnight 4°C

-2. Block endogenous Fc receptors withF (ab) monomeric secondary antibodies

-3. Incubate sections with 1% Triton (in PBS) at room temperature for 30 min to ‘clean’ the tissue.

-4. Use TBS –Tween 20 as a washing buffer. This often gives less background than using PBS-Tween 20.

Further information may be found at:https://www.abcam.com/index.html?pageconfig=resource&rid=11465



Alternately I would suggest using theMouse on Mouse (M.O.M) Polymer IHC Kit, ab127055 which is designed toallow the use ofmouse antibodies on mouse tissueto obtain excellent signal intensities and clean background.(https://www.abcam.com/Mouse-on-Mouse-MOM-Polymer-IHC-Kit-ab127055.html).





I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

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Answer

Thank you for contacting us.

I am sorry to hearthat a customer is 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 attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary.

Of the information the customer provided already, I would like to ask also the two following points, which seem very important to me:

1.) Is the region of glomerular mesangial tissue included in the kidney sections of the samples?

2.) Are the sections from human or mouse kidney?

3.) What different blocking solutions/steps have been used?

I look forward to receiving your reply.

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1-10 of 20 Abreviews or Q&A

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