Anti-Calnexin antibody, rabbit serum
Product code73-026

Size

100 µlStorage

Store 4℃ for short term For long term storage store at -20℃.
Aliquot to avoid repeated freezing and thawing.

Concentration

N/A

Buffer

0.1% sodium azide

PurityRabbit antiserum

ImmunogenC-terminal peptide of mouse Calnexin protein, EDEILNRSPRNRKPRRE,
conjugated with KLH

IsotypeRabbit IgG

Reactivity

Mouse, rat, humanSpecial notes

N/A

Application

1. Western blotting (1/1,000 dilution))
2. Immunoprecipitation (1/100 dilution).
3. Immunohistochemistry (1/100 – 1/1,000 dilution)
4. Immuno-fluorescent staining (1/100~1/300 dilution)

Background

Calcium-binding protein that interacts with newly synthesized glycoproteins in theendoplasmic reticulum. It may act in assisting protein assembly and/or in theretention within the ER of unassembled protein subunits. It seems to play a majorrole in the quality control apparatus of the ER by the retention of incorrectly folded
proteins. Associated with partial T-cell antigen receptor complexes that escape theER of immature thymocytes, it may function as a signaling complex regulating
thymocyte maturation. Additionally it may play a role in receptor-mediatedendocytosis at the synapse.
Molecular mass: 67,278 Da with 591 amino acids.
It undergoes palmitoylation and phosphorylation.

Data Link

uniprot/P35564 mouse calnexin. Gene ID 12330 mouse calnexin.

Please note: All products are FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC
PROCEDURES. NOT FOR MILITARY USE.
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Data Images: 73-026 Anti-Calnexin antibody, rabbit serum

Fig.1 Western blotting analysis of mouse testis extracts of different ages with anti-Calnexin (CANX)
antibody.
20 μg of Triton X-100 extracts from mouse testis was reacted with anti-Calnexin antiserum at 1/1,000
dilution.
Calnexin expression started at birth. (wks) stands for weeks.

Fig.2 Western blotting analysis of lysatess of mouse testis and sperm with anti-Calnexin (CANX)
antibody.
Proteins of the lysates (10 μg) of mouse testis and sperm were separated on SDS-PAGE and blottedto PVDF membrane and reacted with anti-Calnexin antiserum at 1/1,000 dilution. Calnexin bands
migrated to the position corresponding to molecular mass of 90 kDa.

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Fig.3.Immunoprecipitation of Calnexin from mouse testis. One mg of testis lysate (supernatant inlysis buffer containing 10 mM Tris-HCl pH8.0, 50 mM NaCl, 1% protease inhibitor mix) was incubatedwith 2 μl of anti-Calnexin (CANX) antiserum and 50 μl. of protein-A conjugated magnetic beads(MiltenyiBiotec)

andimmunoprecipitatedaccording

totheprotocolofsupplier.

The

immunoprecipitated sample was analyzed by western blotting using the same antibody at 1/1,000dilution.

Fig.4 Western blotting analysis of lysatess of human and mouse cell lines with anti-Calnexin (CANX)
antibody.
Proteins of the lysates (20 μg) of HeLa, MCF7 and NIH3T3 cells were separated on SDS-PAGE andblotted to PVDF membrane and reacted with anti-Calnexin antiserum at 1/1,000 dilution. As second
antibody, goat polyclonal antibody to rabbit IgG conjugated with HRP (ab97051) was used.

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Fig.5. Immunohistochemistry of mouse testis using anti-CANX 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

100 dilution in PBS- 4℃ O/NWashing

PBS-, 5 min, 3times

2nd antibody

1000 dilution, 60 min (Alexa Flour-488 goat anti-rabbit IgG (H&L),#1166843 ; molecular probes)

washing

PBS-, 5 min, 3times

DAPI

1.0μg/mL DAPI in TBS, 10 min

Washing

PBS-, 5 min, 3times

Mount

ImmunoSelect Antifading Mounting Medium (SCR-38447; Dianova)

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Fig.6 Immunofluorescent staining of HeLa cells with anti-Calnexin antibody
Anti-mouse CANXDAPIMerge

1/100 dilution
Reference: This antibody has been described and used in the following publication.
1. Inoue N. et al. Identification and disruption of sperm-specific angiotensin converting enzyme-3(ACE3) in mouse. PubMed PLoS One. 2010 Apr 22;5(4):e10301. doi: 10.1371. Open access. WB, IP,IF
1/300 dilution

wash
PBS-, 2times
fixed
4 % PFA r.t. for 30 min/ 4℃ over nightwash
PBS- 5 min X 3 timespermeabalized 0.25 % Triton X-100/PBS 10 min
wash
PBS- , 5 min X 3 times
blocking
1.5% BSA/PBS, 30 min
1st antibody dilution buffer; 1.5 % BSA/PBS or CanGet signal B
100, 300 or 1000 dilution, 4℃ over night (r.t. for 60 min)wash
PBS- , 5 min X 3 times
2nd antibody 1000 dilution, 60 min (Alex Fluor 488 goat-anti-rabbit IgG (H&L); molecular probes)
wash
PBS- , 5 min X 3 times
DAPI
1.0μg/mL DAPI in TBS, 5 min (X100 stock, 0.1mg/mL)
wash
0.1 % Triton X-100/TBS, 5min
TBS, 5minmount
ImmunoSelect Antifading Mounting Medium (SCR-38447; Dianova)

Reference: This antibody was described in Ref.1 and used in the following publications.
1. Ikawa M. et al. (2001) Calmegin Is Required for Fertilin α/β Heterodimerization and Sperm
Fertility. Dev Biol. 240: 254-61. IP. Open access.
2. Ikawa M. et al. (2011) Calsperin is a testis-specific chaperone required for sperm fertility. J Biol
Chem. 286: 5639-46. WB, IP. Open access.

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