Overview

  • Product name
    Anti-CD59 antibody [MEM-43/5]
    See all CD59 primary antibodies
  • Description
    Mouse monoclonal [MEM-43/5] to CD59
  • Host species
    Mouse
  • Specificity
    CD59 antigen (human). MEM-43/5 reacts with well defined epitope (around L33) and does not compete with MEM-43 and many other CD59 antibodies
  • Tested applications
    Suitable for: ICC/IF, IHC-P, WB, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Thymocytes and T lymphocytes.

  • Epitope
    The antibody MEM-43/5 reacts with well defined epitope around L33 (see Bodian et al)
  • Positive control
    • Flow cyt: blood Jeg3 cell line IF/ICC

Properties

Applications

Our Abpromise guarantee covers the use of ab9183 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 a concentration of 5 µg/ml.
IHC-P Use a concentration of 5 µg/ml.
WB Use a concentration of 1 - 2 µg/ml. Use under non reducing condition. CD59 is GPI-anchored, so we recommend to use a laurylmatoside based lysis buffer or triton base buffer (see Bodian et al; 1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml pepstatin A and 1 mM phenlymethylsulphonyl fluoride in PBS), not NP40.
IP Use at an assay dependent concentration.
Flow Cyt Use a concentration of 1 µg/ml.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

 

Target

  • Function
    Potent inhibitor of the complement membrane attack complex (MAC) action. Acts by binding to the C8 and/or C9 complements of the assembling MAC, thereby preventing incorporation of the multiple copies of C9 required for complete formation of the osmolytic pore. This inhibitor appears to be species-specific. Involved in signal transduction for T-cell activation complexed to a protein tyrosine kinase.
    The soluble form from urine retains its specific complement binding activity, but exhibits greatly reduced ability to inhibit MAC assembly on cell membranes.
  • Involvement in disease
    Defects in CD59 are the cause of CD59 deficiency (CD59D) [MIM:612300].
  • Sequence similarities
    Contains 1 UPAR/Ly6 domain.
  • Post-translational
    modifications
    N- and O-glycosylated. The N-glycosylation mainly consists of a family of biantennary complex-type structures with and without lactosamine extensions and outer arm fucose residues. Also significant amounts of triantennary complexes (22%). Variable sialylation also present in the Asn-43 oligosaccharide. The predominant O-glycans are mono-sialylated forms of the disaccharide, Gal-beta-1,3GalNAc, and their sites of attachment are probably on Thr-76 and Thr-77. The GPI-anchor of soluble urinary CD59 has no inositol-associated phospholipid, but is composed of seven different GPI-anchor variants of one or more monosaccharide units. Major variants contain sialic acid, mannose and glucosamine Sialic acid linked to an N-acetylhexosamine-galactose arm is present in two variants.
    Glycated. Glycation is found in diabetic subjects, but only at minimal levels in nondiabetic subjects. Glycated CD59 lacks MAC-inhibitory function and confers to vascular complications of diabetes.
  • Cellular localization
    Cell membrane. Secreted. Soluble form found in a number of tissues.
  • Information by UniProt
  • Database links
  • Alternative names
    • 16.3A5 antibody
    • 1F5 antibody
    • 1F5 antigen antibody
    • 20 kDa homologous restriction factor antibody
    • CD 59 antibody
    • CD_antigen=CD59 antibody
    • CD59 antibody
    • CD59 antigen antibody
    • CD59 antigen complement regulatory protein antibody
    • CD59 antigen p18 20 antibody
    • CD59 antigen p18-20 (antigen identified by monoclonal antibodies 16.3A5, EJ16, EJ30, EL32 and G344) antibody
    • CD59 glycoprotein antibody
    • CD59 molecule antibody
    • CD59 molecule complement regulatory protein antibody
    • CD59_HUMAN antibody
    • Cd59a antibody
    • Complement regulatory protein antibody
    • EJ16 antibody
    • EJ30 antibody
    • EL32 antibody
    • FLJ38134 antibody
    • FLJ92039 antibody
    • G344 antibody
    • HRF 20 antibody
    • HRF-20 antibody
    • HRF20 antibody
    • Human leukocyte antigen MIC11 antibody
    • Ly 6 like protein antibody
    • Lymphocytic antigen CD59/MEM43 antibody
    • MAC inhibitory protein antibody
    • MAC IP antibody
    • MAC-inhibitory protein antibody
    • MAC-IP antibody
    • MACIF antibody
    • MACIP antibody
    • MEM43 antibody
    • MEM43 antigen antibody
    • Membrane attack complex (MAC) inhibition factor antibody
    • Membrane attack complex inhibition factor antibody
    • Membrane inhibitor of reactive lysis antibody
    • MGC2354 antibody
    • MIC11 antibody
    • MIN1 antibody
    • MIN2 antibody
    • MIN3 antibody
    • MIRL antibody
    • MSK21 antibody
    • p18 20 antibody
    • Protectin antibody
    • Surface antigen recognized by monoclonal antibody 16.3A5 antibody
    • T cell activating protein antibody
    see all

Images

  • IHC image of ab9183 staining CD59 in Human normal placenta formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with EDTA (pH9, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab9183, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  • ICC/IF image of ab9183 stained Jeg3 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 (ab9183, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 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.
  • Overlay histogram showing Jurkat cells stained with ab9183 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab9183, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

References

This product has been referenced in:
  • Kitterer D  et al. Alteration of membrane complement regulators is associated with transporter status in patients on peritoneal dialysis. PLoS One 12:e0177487 (2017). Read more (PubMed: 28542228) »
  • Reales E  et al. The MAL protein is crucial for proper membrane condensation at the ciliary base, which is required for primary cilium elongation. J Cell Sci 128:2261-70 (2015). Read more (PubMed: 25967552) »
See all 7 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Question
Answer

Thank you very much for your interest in ab9183 and ab30532.
As discussed over the phone yesterday, to our knowledge, ab9183 has not been tested in IHC frozen sections. This does not mean that it will not work, simply that we have not tested it ourselves and we have had no reports of our customer using it for this either. Therefore, I can offer a discount off a future purchase if you buy ab9183 now, test it in frozen sections and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of: 1 free primary antibody or the value off you next order.
If you are interested in this offer, please follow these steps:
1. Reply to this e-mail to let me know that you would like to proceed and test ab9183 in frozen sections. I will then send a discount code. This code must be issued before purchasing ab9183 so please wait for my reply before ordering.
2. Purchase ab9183 either by phone, fax, or online (www.abcam.com).
3. Test it in frozen sections.
4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: https://www.abcam.com/abreviews.
5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any product ordered and the discount code is valid for 4 months after issue.
Please remember that submission of the Abreview is sufficient for the discount code to become active.
We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab9183 turns out to be unsuitable for frozen sections, you will still receive the discount on your next purchase after your Abreview has been submitted.
Please let me know if you have any questions about this offer and I would be happy to help you further.
The Terms and Conditions of this offer can be found at:
www.abcam.com/collaborationdiscount
As discussed over the phone I have also had a look into the anti-LDL receptor antibody you were also interested in. Although as you say there have been two negative Abreviews using this product in frozen sections, this is from quite some time ago and we have had no similar reports recently. Please be reassured that if you were to have any problems the product would be covered by the Abpromise and if it did not function as specified on the datasheet you would be entitled to a replacement antibody or a refund if you reported the problem within 6 months. More information on the Abpromise can be found from the following link:
www.abcam.com/abpromise
If I can help in any other way please do let me know.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (placenta)
Specification
placenta
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated
Permeabilization
No

Abcam user community

Verified customer

Submitted Apr 09 2012

Question
Answer

Thank you for calling Abcam.

I am sorry that you were having problems with ab9182 in IHC-P. As we discussed, I am sending you ab9183 as a free of charge replacement and you should have it by 10.30am tomorrow morning. You r new order number is *****.

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

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Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No specific band,just two unspecific bands SAMPLE liver tissue of mice PRIMARY ANTIBODY Ab9183/blocking buffer/1:200-1:1000/over night in +4 degree washing buffer, 5min, 3 times DETECTION METHOD ECL and DAB POSITIVE AND NEGATIVE CONTROLS USED 7402(human hepatoma cell line) protein as positive control ANTIBODY STORAGE CONDITIONS -80℃ SAMPLE PREPARATION 1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml pepstatin A and 1 mM phenlymethylsulphonyl fluoride in PBS boiling water,3 min AMOUNT OF PROTEIN LOADED 40ug/well Loading buffer:No ß-mercaptoethanol or DTT,with SDS ELECTROPHORESIS/GEL CONDITIONS Buffer:25mmol/l Tris Base, 250 mmol/l Glycine, 0.1% SDS Gel Composition:acrylamide and N,N-methylenebisacrylamide, SDS, APS, TEMED Gel percentage:12% TRANSFER AND BLOCKING CONDITIONS Buffer: 14.15g/l Glycine ,3g/l Tris Base, 20% Methanol Time period: 1 hour and 15 minutes Blocking agent:5% non-fat milk SECONDARY ANTIBODY Beijing Zhongshan Golden Bridge Biotechnology Co.,LTD/ Goat anti-mouse/ blocking buffer/1:1500/1 hour washing buffer, 5min, 3 times HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES All steps is the same between 7402 and liver of mouse.The result seems all right for CD59 of 7402,while,there's no specific band for mouse liver but two non-specific bands about 25-30KD,which also appear in the "No Primary" control. Also, beta-actin control is OK.

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Answer

Thank you for your patience. I am sorry to hear that you have been experiencing problems with ab9183 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a free replacement/credit note/refund will be offered. I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. If the suggestions do not prove to be helpful, would you please be so kind to confirm the following items in order to help me better understand the cause of the problem. This product should be able to detect a band at around 19-25kDa. Therefore the bands you are observing above the 25kDa may be multimers. Can you confirm you have reduced and denatured the sample at 95oC for 10 minutes in buffer containing SDS and mercaptoethanol? This will ensure that the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. As a rule of thumb, all of our products need to be run at a reducing condition unless stated otherwise on the datasheet. Please use reducing condition if you have not already done so. From what you mentioned about seeing bands at the 25-30kDa when running without primary antibody, it is obvious that the bands are caused by non-specific binding of the secondary antibody. Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? If possible, please try another goat anti-mouse antibody. Was the lysis buffer you used as strong as RIPA buffer? Please try RIPA buffer as it is the strongest and will ensure that you get all the protein of interest out. I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with details of your order (purchase order number, lot number, contact information, shipping address/purchasing agent). Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund to you.

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Answer

Thank you for taking the time to contact us. I am sorry to hear the customer has difficulty obtaining satisfactory results from this antibody (ab9183). CD59E is a glycolipid-anchored protein, and these anchored membrane proteins tend to form detergent resistant complexes and require addition of other agents to the lysis buffer. I am sorry I am unable to trace further details of the use of laurylmatoside. I can, however, suggest the following reference. This includes a section on the Analysis of Detergent-insoluble complexes. I hope this information should help. Journal of Biological Chemistry 1997, Vol 272 (31) 19242-19247. Signal Transduction via Glycosyl Phosphatidylinositol-anchored Proteins in T Cells Is Inhibited by Lowering Cellular Cholesterol* Thomas M. Stulnig et al. PMID: 9235917 Should the customer have further difficulty, I can suggest they can complete our technical questionaire. We can then look into the details of their procedure and offer assistance. https://www.abcam.com/index.html?section=western&pageconfig=technical&mode=questionaire I hope this information is helpful. Please do not hesitate to contact me again if the customer has any more questions, or has any further difficulties with this product.

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Question
Answer

Thank you for your enquiry. Here is the answer from the originator: "CD59 is small glycoprotein, is SDS-PAGE it migrates about 18-25 KDa due to carbohydrate moiety (if treated with endoglycosidases, it moves faster). It is GPI-anchored, so that the lysis must be ell efficient (like using laurylmatoside, not NP-40 detergent)." Please let me know if I can be of any further assistance.

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Answer

The blocking potential of this antibody has not yet been determined, nor has it's cross-reactivity with porcine CD59.

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