anti-CD52 (Mouse) Antibody, Rabbit Serum

Description

CD52 may play a role in carrying and orienting carbohydrate, as well as having a more specific role. Expressed on lymphohematopoietic tissues, including thymus, spleen, and bone marrow, but not in liver, kidney, and brain.

 

Molecular mass: 7,798 Da with 74 amino acids. In mature form, propeptide is removed, and GPI anchored and glycosylated.

 

Key words: CD52, CAMPATH-1 antigen, Lymphocyte differentiation antigen B7, Cell membrane, GPI-anchor, Glycoprotein.

 

Specifications:

Reactivity: Mouse. Likely to react with rat due to the sequence identity.

Immunogen: Synthetic peptide corresponding to 29-48 amino acids of mouse CD52, C-AASGTNKNSTSTKKTPLKSG, conjugated with KLH

Form: Whole rabbit antiserum added with 0.1% sodium azide.

Validation: Specificity validated with KO mouse (Fig.2)

Storage: Shipped at 4℃ or at -20℃. Upon arrival, spin-down and store at -20℃.

 

Applications:

Western blotting (1/1,000 dilution)

Immunohistchemistry-P (1/100 dilution)

Immunofluorescence staining (1/100 dilution)

 

Database Links:

uniprot/Q64389 Mouse CD52

Gene ID 23833  Mouse CD52

 

Reference: This antibody was described in Ref.1 and used in the following publications.

 

Yamaguchi R et al. (2008) Cd52, known as a major maturation-associated sperm membrane antigen secreted from the epididymis, is not required for fertilization in the mouse. Genes Cells.13:851-61.WB.

 

 

Fig.1 Western blotting analysis of CD52 expression in various tissues with anti-CD52 antibody. Testes, male reproductive ducts and sperm protein were extracted with lysis buffer containing Triton X-100 and subjected to western blot analysis. Western blots containing equal amounts of tissue proteins (30 µg) and sperm protein (10 µg) were reacted with anti-CD52 antibody at 1/1,000 dilution.

 

 

 

Fig.2. Western blot analysis of CD52 in cauda epididymal lysates and sperm lysates of wild type and CD52 deficient mice.  Cd52^+/+ (+/+), Cd52^+/– (+/–), and Cd52^−/–(–/–).

Cauda epididymis and sperm from vas deferens were lysed in lysis buffer containing 1% TritonX-100. Proteins (30 μg for cauda epididimis and 10 μg for sperm) were analyzed by western blotting with anti-CD52 antibody at 1/1,000 dilution.

 

 

Fig.3. Western blot analysis of CD52 in lysates of mouse testis and sperm with anti-CD52 antibody. Proteins in the lysates (10 μg) are separated on SDS-PAGE (10-20% gradient) and electro-blotted to PVDF membrane. The membrane was reacted with anti-CD52 antibody at 1/1,000 dilution. As the second antibody, anti-rabbit IgG antibody conjugated with HRP (ab97051) was used at 1/10,000 dilution. The numbers on the right are positions of protein size markers shown in kDa.

 

 

Fig.4. Immunohistochemistry of mouse testis using anti-CD52 antibody. Formalin-fixed and paraffin-embedded mouse testis Deparaffinization by LemosolRA (#122-03991, Wako, Osaka)

Rehydration 100% Et-OH, 95%, 90%, 70%, DW

Antigen retrieval Histo/Zyme (Cat.# k046; Diagnostic BioSystems)

Washing   PBST (0.25% triton X-100/PBS-)

Blocking 10 % FBS / PBST 30 min

1st antibody 1/100 dilution in PBS- 4℃ O/N

Washing PBS-

2nd antibody 1,000 dilution, 60 min (AF-488 goat anti-rabbit IgG (H&L)

Washing   PBS- 5 min, 3 times

DAPI 1.0μg/mL DAPI in TBS 10 min

Mount ImmunoSelect Antifading Mounting Medium (SCR-38447; Dianova)

 

 

Fig. 5 Immunofluorescence staining of CD52 in NIH3T3 cells with anti-CD52 antibody. The cells were fixed in 4% paraaformaldehyde overnight.

Permeabilization in 0.25% Triton X-100/PBS for 10 min

Blocking in 1.5% BSA/PBS for 30 min

1st antibodies diluted 1/100 by blocking buffer and incubated over night

2nd antibody,goat, anti-mouse IgG conjugated withAlex 488 (1/1000 dilution).

Nuclei were stained with DAPI.