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Purified Mouse Anti-Calnexin
Purified Mouse Anti-Calnexin

Western blot analysis for Calnexin on a HeLa cell lysate (Human cervical epitheloid carcinoma; ATCC CCL-2). Lane 1: 1:1000, lane 2: 1:2000, lane 3: 1:4000 dilution of the Mouse Anti-Calnexin antibody.

Purified Mouse Anti-Calnexin

Immunofluorescence staining of A549 (Human lung carcinoma; ATCC CCL-185) cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were stained using the Perm Buffer III protocol and the Mouse Anti-Calnexin antibody.  The second step reagent was a FITC Goat Anti-Mouse Ig polyclonal antibody (Cat. No. 554001).  Images were taken on a BD Pathway™ 855 bioimaging system using a 20x objective.  This antibody also stained HeLa (ATCC CCL-2) and U-2 OS (ATCC HTB-96) cells with either the Triton™ X-100 and or Perm Buffer III protocols (see Recommended Assay Procedure).

Western blot analysis for Calnexin on a HeLa cell lysate (Human cervical epitheloid carcinoma; ATCC CCL-2). Lane 1: 1:1000, lane 2: 1:2000, lane 3: 1:4000 dilution of the Mouse Anti-Calnexin antibody.

Immunofluorescence staining of A549 (Human lung carcinoma; ATCC CCL-185) cells. Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were stained using the Perm Buffer III protocol and the Mouse Anti-Calnexin antibody.  The second step reagent was a FITC Goat Anti-Mouse Ig polyclonal antibody (Cat. No. 554001).  Images were taken on a BD Pathway™ 855 bioimaging system using a 20x objective.  This antibody also stained HeLa (ATCC CCL-2) and U-2 OS (ATCC HTB-96) cells with either the Triton™ X-100 and or Perm Buffer III protocols (see Recommended Assay Procedure).

Product Details
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BD Transduction Laboratories™
Human (QC Testing), Mouse, Rat, Dog (Tested in Development)
Mouse IgG1
Human Calnexin aa. 116-301
Western blot (Routinely Tested), Bioimaging, Immunohistochemistry, Immunoprecipitation (Tested During Development)
90 kDa
250 µg/ml
AB_397883
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at -20°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Recommended Assay Procedures

Bioimaging:  For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp.  An abbreviated protocol is listed below:

1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.

2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well.  Incubate for 10 minutes at room temperature (RT).

3. Remove the fixative from the wells, and permeabilize the cells using either BD Phosflow™ Perm Bufer III, 90% methanol or Triton™ X-100:

a. Add 100 μl of -20°C chilled Perm Buffer III (Cat. No. 558050) or 90% methanol to each well and incubate for 5 minutes at RT.

     OR

b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.

4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.

5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.

6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.

7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.

8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.

9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.

10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

11. View and analyze the cells on an appropriate imaging instrument.

Western blot:  For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
  3. Triton is a trademark of the Dow Chemical Company.
  4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
610523 Rev. 3
Antibody Details
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37/Calnexin

Calnexin is a 90 kDa integral membrane protein located primarily in the ER.  It contains a long N-terminal calcium-binding domain that extends into the lumen of the ER and a short, acidic cytosolic domain.  Calnexin associates with several cell surface proteins as they pass through the ER.  Since it also forms complexes with other resident ER proteins involved in the Ca2+-dependent retention of proteins, calnexin may act as a chaperone.  The amino acid sequence of calnexin is highly conserved among species and is similar in sequence to calreticulin, another Ca2+-binding protein found in the ER.  Neither calnexin nor calreticulin contain the calcium-binding motif, known as the "E-F hand", found in the calmodulin family of proteins.

610523 Rev. 3
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
610523 Rev.3

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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.