Overview

  • Product name
  • Description
    Rabbit polyclonal to C5b-9
  • Host species
    Rabbit
  • Specificity
    This antibody is monospecific for C5b-9 complex in purified form or present in cobra venom factor activated human serum. There is no reactivity vs. non-activated normal human serum or plasma
  • Tested applications
    Suitable for: IHC-Fr, IHC-P, IHC-FoFr, RID, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Full length native protein (purified) corresponding to Human C5b-9. Purified human SC5b-9 complex.

Properties

Applications

Our Abpromise guarantee covers the use of ab55811 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-Fr Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
IHC-FoFr Use at an assay dependent concentration.
RID Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.

Target

  • Relevance
    Activation of the complement system plays a key role in normal inflammatory response to injury but may cause substantial injury when activated inappropriately. The complement system is activated either through the classical (antibody induced) or the alternative (microbial surface, polysacharride induced) pathway, both leading to the formation of the C5b9 complex. Fluid phase binding of the multifunctional glycoprotein S protein (vitronectin) to C5b9 leads to the formation of a cytolytically inactive complex, SC5b9, which is unable to attach to cells.
  • Cellular localization
    Secreted
  • Database links
  • Alternative names
    • C5 antibody
    • c5b 9 antibody
    • C6 antibody
    • C7 antibody
    • C8 antibody
    • C9 antibody
    • Complement component 5 antibody
    • Complement component 6 antibody
    • Complement component 7 antibody
    • Complement component 8 antibody
    • Complement component 9 antibody
    see all

Images

  • ab55811 staining C5b-9 in human liver.
    Left panel: with primary antibody at 4 ug/ml. Right panel: isotype control.
    Sections were stained using an automated system (DAKO Autostainer Plus ), at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
  • ICC/IF image of ab55811 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab55811, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • Jeon H  et al. Activation of the complement system in an osteosarcoma cell line promotes angiogenesis through enhanced production of growth factors. Sci Rep 8:5415 (2018). ELISA, ICC/IF . Read more (PubMed: 29615744) »
  • Tougan T  et al. Molecular Camouflage of Plasmodium falciparum Merozoites by Binding of Host Vitronectin to P47 Fragment of SERA5. Sci Rep 8:5052 (2018). Read more (PubMed: 29567995) »
See all 34 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Loading amount
80 µg
Gel Running Conditions
Non-reduced Denaturing
Sample
Mouse Tissue lysate - whole (Brain)
Specification
Brain
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jun 23 2014

Question
Answer

Thank you for contacting us.

The datasheet pdfs can be downloaded from Abcam website.

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

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Answer

Thank you for contacting us.

I am sorry that this antibody did not perform as stated on the datasheet. As requested, I have asked our Finance department to issue a credit note for you.

Credit note ID: ####


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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Question

Order Details
Antibody code: ab55811

Lot number: GR81263-4

Purchase order number CCE 3816178
or preferably Abcam order number: 1119805


General Information
Antibody storage conditions (temperature/reconstitution etc)

Aliquots at -20 degrees


Description of the problem (high background, low signal, non-specific satining etc.)
No specific staining, comparable to "no primary antibody" control.


Sample (Species/Tissue/Cell Type/Cell Line etc.)

PFA fixed Paraffin embedded mouse kidney from MRL/lpr mice andBalb/C mice infected with E. coli O157:H7


Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
4% PFA in phosphate buffer

Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Boiling 20 min in Citrate buffer pH: 6.0

Boiling 20min in Tris buffer pH: 9.0 (10mM Tris 1mM EDTA 0,05% Tween 20)


Permeabilization step
No

Blocking conditions (Buffer/time period, Blocking agent etc.)
TBS with 1% BSA 10% Normal Goat serum 1h RT

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Abcam ab55811 antibody in TBS with 1% BSAdilution from 5ug/mL to 50ug/mLover night.

Wash 2x5min in TBS with triton x-100

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Invitrogen Alexa fluor 488 Goat anti rabbit in TBS with 1% BSAdilution1:400

DAKO EnVision System HPR labelled polymer Anti-Rabbit

Detection method
IF microscope with Alexa 488
IHC DAKO DAB+ haematoxilin

Positive and negative controls used (please specify)
Negative control tissue is kidneys from non infected Balb/C mice

Optimization attempts (problem solving)
How many times have you tried the IHC?
8


Have you run a "No Primary" control?
Yes

Do you obtain the same results every time?
Yes


What steps have you altered?
Antigen retrieval step

Dilutions of primary and secondary antibodies.

Different tissues from mice MRL/lpr that has stained positive in another report for C5b-9 using another antibody
and Balb/C mice infected withE. coli O157:H7
Additional Notes
Reference paper: Morigi 2011The Journal of ImmunologyJuly 1, 2011vol. 187no. 1172-180

We would appreciate if you are also able to provide and image which woudl help us to assess the results

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Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will provide us with vital information for our monitoring of product quality

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been succesful. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably recieved a bad vial.

I apologise for the inconvenience and am pleased to offer you a free of charge replacement or credit note in compensation.

In addition, I can suggest you may like to consider the following suggestions as a check for future experiments:

1. The time for antigen retreival can sometimes require optimization, and a shorter can be better. I can recommend to try 2, 5, 10 minutes.

2. We recommend not to mix blocknig agents in one experiment. This can sometimes affect the results. Try 10% Serum only.

3. Wash 4 times rather than 2 times at each wash step.

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 taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for IHC-P, IHC-Frand human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily. Once we have this information, we can review the protocol to see if there are any further recommendations we can make.

I would appreciate if you could also provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.



Order Details
Antibody code:

Lot number:


Purchase order number
or preferably Abcam order number:


General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, low signal, non-specific satining etc.)

Sample (Species/Tissue/Cell Type/Cell Line etc.)


Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)


Antigen retrieval (Enzymatic method, Heat mediated technique etc.)


Permeabilization step


Blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method


Positive and negative controls used (please specify)


Optimization attempts (problem solving)
How many times have you tried the IHC?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No


What steps have you altered?


Additional Notes


We would appreciate if you are also able to provide and image which woudl help us to assess the results

Read More

Answer

Thank you for your response and updates.

Wehope the second vial will work as it is expected, and please do letus know how you are getting on with this product.

If you need any further assistance, please do not hesitate to contact our Scientific Support Team.

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Question
Answer

Thank you for your enquiry and your interest. My colleague has passed your enquiry to the Scientific Team.

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.

If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

Could you provide some further details of the protocol used and confirm the batch and the Abcam Order Number (PON). It would be much appreciated if you could attach an image to the response.

I look forward to receiving your reply.

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Answer

Thank you for contacting Abcam.

We have not tested ab55811 on rat tissue that does mean to say that it will not work, just that we have not tried it.

We are basing reactivity towards both human and mouse tissue based on in house testing of the product (mainly IF and IHC) and on publications that have used the product (links to these publications are found on the webpage for this product).

If there is anything else I can help you with, please let me know.

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Answer

Thank you for your enquiry. The reason that the molecular weight is different could be that the C5-b-9 complex degrades under proteolysis and therefore on a western blot, we are only seeing degradation products. In this case, it is the phosphorylated C9 which we are seeing. An example can be seen in the WB image for ab83076. "C5 is cleaved into C5 alpha and C5 beta chains, joined by a disulphide bond. C5 alpha is cleaved leaving C5a (anaphylatoxin, which is released) and C5b (alpha’ + beta chains). C5b then binds sequentially to C6, C7, C8 and C9 to form the Membrane attach complex (MAC), also known as C5b-9. In rat urine, C5b-9 has been shown to be degraded presumably by proteolysis (Kerjaschkl et al., 1989). We hypothesise that the bands at 30- and 55-kDa correspond to degradation products." I hope this information will be helpful.  

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Answer

Thank you for your inquiry. I have contacted the originator of the ab55811 product and they have confirmed that this product was used in the following publication: Schafer, H., et al. 1986 (J. Immunol. 137, 1945), which suggests that this antibody recognizes C5b-9 "at membranes". The authors used it to detect C5b-9 deposits at the cell membrane (Fig 3b of the article). http://jimmunol.org/cgi/content/abstract/137/6/1945 Please rest assured that all of our products are guaranteed to work in species and application as stated on the datasheet. Should you not get expected results, then please inform us within the guarantee period of 120 days. We will help troubleshoot the experiment and if the product is indeed faulty, we will be more than happy to offer a free replacement or refund. I hope our Abpromise guarantee will give you the assurance you need to use this product in your research.

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

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