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BD Transduction Laboratories™ Purified Mouse Anti-Calretinin
Clone 34/Calretinin (RUO)
Western blot analysis of Calretinin on a rat cerebrum lysate. Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the Mouse Anti-Calretinin antibody.
Immunofluorescent staining of A549 (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 alcohol perm protocol and the Mouse Anti-Calretinin antibody. The second step reagent was FITC goat anti mouse Ig (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) cells using the alcohol perm protocol and A549, HeLa and U-2 OS (ATCC HTB-96) cells using the Triton™ X-100 fix/perm protocol (see Recommended Assay Procedure).
Western blot analysis of Calretinin on a rat cerebrum lysate. Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of the Mouse Anti-Calretinin antibody.
Immunofluorescent staining of A549 (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 alcohol perm protocol and the Mouse Anti-Calretinin antibody. The second step reagent was FITC goat anti mouse Ig (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) cells using the alcohol perm protocol and A549, HeLa and U-2 OS (ATCC HTB-96) cells using the Triton™ X-100 fix/perm protocol (see Recommended Assay Procedure).
Product Details
BD Transduction Laboratories™
Rat (QC Testing), Human, Mouse (Tested in Development)
Mouse IgG1
Rat Calretinin aa. 38-151
Western blot (Routinely Tested), Bioimaging (Tested During Development)
29 kDa
250 µg/ml
AB_398225
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
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 Perm Buffer III, 90% methanol, or Triton™ X-100:
a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) 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.
Bioimaging: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp
Western blot: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/monoclonal_anti.jsp
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- 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.
- Triton is a trademark of the Dow Chemical Company.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 ML
Cat No.
554656
Fixation Buffer RUO
Size
100 ML
Cat No.
554655
Perm Buffer III RUO
Size
125 ML
Cat No.
558050
Cat No.
353219
HRP Goat Anti-Mouse Ig RUO
Size
1 ML
Cat No.
554002
FITC Goat Anti-Mouse Ig RUO
Size
0.5 mg
Cat No.
554001
610908 Rev. 3
Antibody Details
34/Calretinin
A low level of intracellular free Ca2+ is essential for a variety of cellular functions and is mediated by the sequestration of Ca2+ in the ER, by the action of plasma membrane Ca2+ pumps, and by Ca2+ binding proteins. The Ca2+ binding protein, Calretinin, is a member of the calmodulin superfamily whose members promote calcium homeostasis by acting as buffers of intracellular free Ca2+. Although Calretinin is expressed in certain populations of neurons throughout the central and peripheral nervous systems, it is primarily expressed in the olfactory bulb and auditory pathways. Calretinin most closely resembles calbindin D28k, another member of the calmodulin family. These two proteins share 58% amino acid identity and contain six EF-hand Ca2+ binding motifs. Excess Calretinin or calbindin results in decreased transcription from PCE1, a calcium responsive element in the calmodulin II promoter. This supports the role of Calretinin as a regulator of intracellular free Ca2+ levels. Furthermore, truncated forms of Calretinin have been found in certain tumor cells, but are absent from normal cells which express wild type Calretinin. Thus, these isoforms may have a role in tumorigenesis.
610908 Rev. 3
Format Details
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
610908 Rev.3
Citations & References
View product citations for antibody "610908" on CiteAb
Development References (4)
-
Arnold DB, Heintz N. A calcium responsive element that regulates expression of two calcium binding proteins in Purkinje cells. Proc Natl Acad Sci U S A. 1997; 94(16):8842-8847. (Biology). View Reference
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Parmentier M, Lefort A. Structure of the human brain calcium-binding protein calretinin and its expression in bacteria. Eur J Biochem. 1991; 196(1):79-85. (Biology). View Reference
-
Schurmans S, Schiffmann SN, Gurden H, et al. Impaired long-term potentiation induction in dentate gyrus of calretinin-deficient mice. Proc Natl Acad Sci U S A. 1997; 94(19):10415-10420. (Biology). View Reference
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Schwaller B, Durussel I, Jermann D, Herrmann B, Cox JA. Comparison of the Ca2+-binding properties of human recombinant calretinin-22k and calretinin. J Biol Chem. 1997; 272(47):29663-29671. (Biology). View Reference
610908 Rev. 3
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
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.