Overview

  • Product name
    Anti-CD4 antibody [10C12]
    See all CD4 primary antibodies
  • Description
    Mouse monoclonal [10C12] to CD4
  • Host species
    Mouse
  • Tested applications
    Suitable for: ELISA, WB, ICC/IF, Blockingmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rabbit, Cat, Dog, Pig, Chimpanzee, Monkey
  • Immunogen

    Synthetic peptide: LS EKKTCQCPHR FQKTCSPI, corresponding to C terminal amino acids 439-458 of Human CD4.

  • Epitope
    This antibody recognizes an epitope within amino acids 414-433 of the cytoplasmic tail region of human CD4.
  • Positive control
    • Jurkat and H9 cell lysates for ELISA, and CEM cells on glass slides for IF.

Properties

Applications

Our Abpromise guarantee covers the use of ab25804 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
ELISA Use a concentration of 10 - 20 µg/ml.
WB Use a concentration of 5 - 10 µg/ml. Predicted molecular weight: 51 kDa.
ICC/IF Use at an assay dependent concentration. PubMed: 20929345
Blocking Use at an assay dependent concentration. PubMed: 22745469

Target

  • Function
    Accessory protein for MHC class-II antigen/T-cell receptor interaction. May regulate T-cell activation. Induces the aggregation of lipid rafts.
  • Sequence similarities
    Contains 3 Ig-like C2-type (immunoglobulin-like) domains.
    Contains 1 Ig-like V-type (immunoglobulin-like) domain.
  • Post-translational
    modifications
    Palmitoylation and association with LCK contribute to the enrichment of CD4 in lipid rafts.
  • Cellular localization
    Cell membrane. Localizes to lipid rafts. Removed from plasma membrane by HIV-1 Nef protein that increases clathrin-dependent endocytosis of this antigen to target it to lysosomal degradation. Cell surface expression is also down-modulated by HIV-1 Envelope polyprotein gp160 that interacts with, and sequesters CD4 in the endoplasmic reticulum.
  • Information by UniProt
  • Database links
  • Alternative names
    • CD 4 antibody
    • CD4 (L3T4) antibody
    • CD4 antibody
    • CD4 antigen (p55) antibody
    • CD4 antigen antibody
    • CD4 molecule antibody
    • CD4 receptor antibody
    • CD4+ Lymphocyte deficiency, included antibody
    • CD4_HUMAN antibody
    • CD4mut antibody
    • L3T4 antibody
    • Leu3 antibody
    • Ly-4 antibody
    • Lymphocyte antigen CD4 antibody
    • MGC165891 antibody
    • OTTHUMP00000238897 antibody
    • p55 antibody
    • T cell antigen T4 antibody
    • T cell antigen T4/LEU3 antibody
    • T cell differentiation antigen L3T4 antibody
    • T cell OKT4 deficiency, included antibody
    • T cell surface antigen T4/Leu 3 antibody
    • T cell surface antigen T4/Leu3 antibody
    • T cell surface glycoprotein CD4 antibody
    • T-cell surface antigen T4/Leu-3 antibody
    • T-cell surface glycoprotein CD4 antibody
    • W3/25 antibody
    • W3/25 antigen antibody
    see all

References

This product has been referenced in:
  • Chae JJ  et al. Nictitating membrane fixation improves stability of the contact lens on the animal corneal surface. PLoS One 13:e0194795 (2018). Read more (PubMed: 29584737) »
  • Wongsena W  et al. Melatonin suppresses eosinophils and Th17 cells in hamsters treated with a combination of human liver fluke infection and a chemical carcinogen. Pharmacol Rep 70:98-105 (2018). Read more (PubMed: 29331794) »
See all 6 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Bos taurus Tissue lysate - whole (Spleen)
Gel Running Conditions
Reduced Denaturing (12%)
Loading amount
45 µg
Treatment
Noninfected controls and virally infected samples
Specification
Spleen
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 24°C

Dr. Katie Knapek

Verified customer

Submitted Mar 22 2018

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (small intestine)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Sodium Citrate Buffer
Permeabilization
No
Specification
small intestine
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Formaldehyde

Dr. Hsiao-Ying chiu

Verified customer

Submitted Dec 22 2014

Answer

Vielen Dank für Ihre Antwort und für diese weiteren Informationen.

Es tut mir leid zu hören, dass unsere Vorschläge in diesem Fall nicht zu einer Verbesserung Ihrer Ergebnisse geführt haben. Ich weiß, dass Sie viel Zeit für diese Experimente verwendet haben und möchte Ihnen daherein Ersatzröhrchen (auch von einem alternativen Antikörper möglich)oder einen Gutschein als Ersatz anbieten.

Würden Sie mir bitte mitteilen, welche Alternative Sie bevorzugen?

Vielen Dank für Ihre Kooperation und Hilfsbereitschaft. Ich freue mich bald wieder von Ihnen zu hören.

Read More

Answer

Thank you for contacting Abcam and for your interest in our products.

Regarding the CD4 antibody ab25804, based on a BLAST of the immunogen, there is no homology with rat, therefore I would not recommend this antibody to use with rat samples. As an alternative, I would like to recommend ab33775. Although it is not tested for ICC/IF and therefore not covered by our Abpromise, I am happy to offer a testing discount in which you purchase the antibody, test it in ICC/IF and submit your results in the form of an Abreview. Once the review has been submitted, the code I issue can be redeemed for a free primary antibody on a future purchase. Please see the terms and conditions for additional information: www.abcam.com/collaborationdiscount.

Regarding ab21029 (T cell Receptor V gamma 3), this antibody is not validated for rat and the epitope has not been mapped. Therefore, we do not know if the antibody will react, but there is 84% overall homology between the mouse and rat proteins so there is a chance it might work. The same testing discount above would apply.

Finally, ab6201 (BDNF), is tested and guaranteed to work with rat samples in ICC/IF.

I hope this information is helpful. If you are interested in learning more about the testing discount, or if you would like to test ab21029 and ab33775, please reply to this email and I will issue the discount codes. Please do not hesitate to contact me if you have any additional questions.

Read More

Answer

Thank you for your email.
I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx with ab846.
To check the status of the order please contact our Customer Service team and reference this number.
Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.
I wish you the best of luck with your research.

Read More

Question

Thank you very much indeed for your response.


PPARA staining:

1- According to publications, it is expected that the nuclei will stain faintly because of the ongoing chronic inflammation. But in healthy subjects one will see stronger staining in the nuclei. I am planning to stain BAL cytospin slides from healthy subjects. If this will show more staining of the nuclei so I can say that the permeablisation is working. If I will get the same result as in the patient group. I need to optimize my procedure as you already mentioned.

2- I would prefer isotype control antibody that compares with the primary. Which one will you recommend?

CD4 Staining:

1- I have tested several methods for fixation: I have testes simple (only CD4) and double staining (CD4 and PPARA simultaneously)

a. Methanol fixation 10 min in – 20 C

b. Folmaldehyde 4% 15 min on Ice and after that Reiterating, quenching buffer. This method is the best.

2- For permeabilisation: Saponin but I have also tested Triton and Tween 20 ( AbCam protocol and concentrations). Saponin and was used in several publications for staining CD4 T cells.

3- Blocking: I have tested several methods: 10% serum from the species that the secondary antibody was raised in or fish serum. Fish serum gave the best result.

4- Concentration of primary Anti-CD4 antibody [10C12] (ab25804) 10 ug/ml

5- Concentration of Rat monoclonal secondary Ab to Mouse IgG1 (PE) 0.1 ug/60 000 cells.

6- My college used also another method (citrate + TX-100) but the staining did not work for CD4 or PPARA

Read More
Answer

Thank you for your reply.
For PPAR alpha, I would recommend ab27478 as an isotype control.
https://www.abcam.com/Rabbit-polyclonal-IgG-ab27478.html

As for the CD4 antibody, your protocol seems to be fine. The immunogen sequence is located in the cytoplasm and thus requires permeabilization as you are doing.
You have tried several troubleshooting steps, so I have no additional tips. It might just be that the vial or the particular lot of the CD4 antibody is not working as expected. I am very sorry about that and can offer you a free of charge replacement with another CD4 antibody (ab846 or ab1246). Alternatively, a credit or refund can be arranged.
Please let me know which option would be best for you.
I look forward to hear back.

Read More

Answer

Thank you for contacting us.
To answer your questions:
1. Yes, a negative control can be ommiting the primary. Other options could be using an isotype control antibody that compares with the priamry, or the blocking peptide. There are various options, but it is up to each reseracher what they prefer to use.
PPAR alpha should stain the nucleus, and your PPAR alpha data do not seem to show that - from what I can see. The DAPI stain seems to dominate the image, and the green from Dylight 488 is difficult to see - which should be also in the nucleus. You might want to increase the amount of antibody being used (for the secondary, but maybe also the primary), or switch to a red color which would be more easy to see in contrast with the blue (if possible). Using less DAPI could help as well.
You also might want to use a stronger detergent than saponin (e.g. NP40 or TX-100) in order to permeabilize the nuclear membrane).
How long did you permeabilize for with 4%PFA?

2) As for the CD4 antibody, please let me what protocol you used for the double staining including antibody dilutions, sequence of staining, fixation step, blocking step etc.
I look forward to hear back from you.

Read More

Answer

Thank you for your inquiry. Unfortunately, we do not offer an antibody that is tested and guaranteed for rabbit and IHC-P. We do not know if ab25804 is suitable for IHC-P. I would like to offer you a testing discount for this, but testing two unknowns (rabbit and IHC-P)at the same time might prove tricky. Would it be possible for your to also test this in the tested species? It would also be possible to choose an antibody that is already tested for IHC-P and we know that it works with paraffin sections and then only test this antibody in rabbit. Which would only be one variable to test. To find out more about our testing discount program please follow this link: https://www.abcam.com/index.html?pageconfig=resource&rid=11998&viapagetrap=collaborationdiscount Please let me know how you would like to proceed. I am looking forward to your answer.

Read More

Question
Answer

Thank you for calling Abcam. I have continued to look through our catalogue for antibodies for CD4 and CD19, that are guaranteed to work in ICC/IF on mouse tissue. I have not found anything that is guaranteed to work in both your requirements, the best I can find are: - ab25804 (CD4) - ab25232 (CD19) If there is anything else I can help you with, please let me know.

Read More

Question
Answer

Thank you for calling Abcam on Friday. For CD4, I have found the following set Protein: ab111463, fragment from 29-390. Antibodies: ab67480, aa7****                      ab51312, aa******* I know that the antibodies bind very close to each other, but they were the only two that I could find that I was confident that the epitope region was in ab111463 and that the epitope regions did not overlap. For CD8, I am still trying to find two suitable antibodies and I will let you know when I have found a suitable pair.    

Read More

1-10 of 12 Abreviews or Q&A

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