Calnexin Antibody - BSA Free

Novus Biologicals | Catalog # NB100-1974

Novus Biologicals
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Key Product Details

Species Reactivity

Validated:

Human, Mouse, Rat, Hamster, Zebrafish

Cited:

Human, Mouse, Fish - Danio rerio (Zebrafish), Hamster

Predicted:

Canine (90%), Orangutan (93%). Backed by our 100% Guarantee.

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Simple Western, Immunoprecipitation

Cited:

Western Blot, Flow Cytometry, Immunocytochemistry/ Immunofluorescence

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
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Product Specifications

Immunogen

A synthetic peptide made to a C-terminal portion of the rat Calnexin protein (between residues 550-591) [Uniprot: P35565]

Reactivity Notes

Hamster reactivity reported in the scientific literature (PMID: 23760268). Use in Zebrafish reported in scientific literature (PMID:23049555).

Localization

Endoplasmic reticulum

Marker

Endoplasmic Reticulum Membrane Marker

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

97 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for Calnexin Antibody - BSA Free

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974]

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974]

Immunocytochemistry/Immunofluorescence: Calnexin Antibody [NB100-1974] - HeLa cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.05% Triton X-100 in PBS for 5 minutes. The cells were incubated with (NB100-1974) at 1 ug/ml overnight at 4C and detected with an anti-rabbit DyLight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.
Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974]

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974]

Immunocytochemistry/Immunofluorescence: Calnexin Antibody [NB100-1974] - NIH3T3 cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.05% Triton X-100 in PBS for 5 minutes. The cells were incubated with (NB100-1974) at 1 ug/ml overnight at 4C and detected with an anti-rabbit DyLight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.
Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974]

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974]

Immunocytochemistry/Immunofluorescence: Calnexin Antibody [NB100-1974] - Rat FR cells were fixed in 4% paraformaldehyde for 10 minutes and permeabilized in 0.05% Triton X-100 in PBS for 5 minutes. The cells were incubated with (NB100-1974) at 1 ug/ml overnight at 4C and detected with an anti-rabbit DyLight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.
Western Blot: Calnexin AntibodyBSA Free [NB100-1974]

Western Blot: Calnexin AntibodyBSA Free [NB100-1974]

Calnexin-Antibody-Western-Blot-NB100-1974-img0010.jpg
Immunohistochemistry: Calnexin Antibody - BSA Free [NB100-1974]

Immunohistochemistry: Calnexin Antibody - BSA Free [NB100-1974]

Immunohistochemistry: Calnexin Antibody [NB100-1974] - Analysis of Calnexin in mouse bladder using DAB with hematoxylin counterstain.
Flow Cytometry: Calnexin Antibody - BSA Free [NB100-1974]

Flow Cytometry: Calnexin Antibody - BSA Free [NB100-1974]

Flow Cytometry: Calnexin Antibody [NB100-1974] - An intracellular stain was performed on rat FR cells with Calnexin Antibody NB100-1974 (blue) and a matched isotype control NBP2-24891 (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 1.0 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Dylight 550 (SA5-10033, Thermo Fisher).
Western Blot: Calnexin AntibodyBSA Free [NB100-1974]

Western Blot: Calnexin AntibodyBSA Free [NB100-1974]

Western Blot: Calnexin Antibody [NB100-1974] - Analysis of Calnexin in HeLa whole cell lysate.
Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974]

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974]

Calnexin-Antibody-Immunocytochemistry-Immunofluorescence-NB100-1974-img0011.jpg
Flow Cytometry: Calnexin Antibody - BSA Free [NB100-1974]

Flow Cytometry: Calnexin Antibody - BSA Free [NB100-1974]

Flow Cytometry: Calnexin Antibody [NB100-1974] - Analysis of Alexa Fluor (R) 488 conjugate of NB100-1974. An intracellular stain was performed on Jurkat cells with Calnexin antibody NB100-1974AF488 (blue) and a matched isotype control NBP2-24893AF488 (orange).
Flow Cytometry: Calnexin Antibody - BSA Free [NB100-1974]

Flow Cytometry: Calnexin Antibody - BSA Free [NB100-1974]

Flow Cytometry: Calnexin Antibody [NB100-1974] - An intracellular stain was performed on HeLa cells with Calnexin antibody NB100-1974AF488 (blue) and a matched isotype control NBP2-24893AF488 (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 488. Image using the Alexa Fluor 488 form of this antibody.
Flow Cytometry: Calnexin Antibody - BSA Free [NB100-1974]

Flow Cytometry: Calnexin Antibody - BSA Free [NB100-1974]

Flow Cytometry: Calnexin Antibody [NB100-1974] - An intracellular stain was performed on Daudi cells with Calnexin Antibody NB100-1974 (blue) and a matched isotype control NBP2-24891 (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 1.0 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Dylight 550 (SA5-10033, Thermo Fisher).
Flow Cytometry: Calnexin Antibody - BSA Free [NB100-1974]

Flow Cytometry: Calnexin Antibody - BSA Free [NB100-1974]

Flow Cytometry: Calnexin Antibody [NB100-1974] - An intracellular stain was performed on NIH3T3 cells with Calnexin Antibody NB100-1974 (blue) and a matched isotype control NBP2-24891 (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 1.0 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Dylight 550 (SA5-10033, Thermo Fisher).
Simple Western: Calnexin AntibodyBSA Free [NB100-1974]

Simple Western: Calnexin AntibodyBSA Free [NB100-1974]

Simple Western: Calnexin Antibody [NB100-1974] - Image shows a specific band for Calnexin in 0.1 mg/mL of HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.
Calnexin Antibody - BSA Free

Calnexin in U-2 OS Human Cell Line.

Calnexin was detected in immersion fixed U-2 OS human osteosarcoma cell line using Rabbit anti-Calnexin Affinity Purified Polyclonal Antibody conjugated to FITC (Catalog # NB100-1974F) (green) at 10 µg/mL overnight at 4C. Cells were counterstained with DAPI (blue). Cells were imaged using a 100X objective and digitally deconvolved.
Calnexin Antibody - BSA Free

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974] -

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974] - Zebrafish Vwf forms pseudo-Weibel-Palade bodies (pseudo-WPBs) in mammalian cell culture. pVWF/Myc-HIS (human VWF, (a–c)) or pzVwf/Myc-HIS (zebrafish Vwf, (d–i)) plasmids were transfected into HEK293T cells. Anti-Myc antibody conjugated to Alexa Fluor 488 (green channel, (a, d, g)) was used for detection & anti-calnexin antibody conjugated to Alexa Fluor 594 (red channel, (b, e, h)) labeled endoplasmic reticulum (ER). Both constructs demonstrate formation of elongated Myc positive & ER negative structures (absence of yellow signal in the merged panels, (c, f, i)) characteristic of pseudo-WPBs (examples are indicated in (a, d), & (g) by arrowheads). Scale bars, 2.5 μm. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/23049555), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
Calnexin Antibody - BSA Free

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974] -

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974] - Zebrafish Vwf forms pseudo-Weibel-Palade bodies (pseudo-WPBs) in mammalian cell culture. pVWF/Myc-HIS (human VWF, (a–c)) or pzVwf/Myc-HIS (zebrafish Vwf, (d–i)) plasmids were transfected into HEK293T cells. Anti-Myc antibody conjugated to Alexa Fluor 488 (green channel, (a, d, g)) was used for detection & anti-calnexin antibody conjugated to Alexa Fluor 594 (red channel, (b, e, h)) labeled endoplasmic reticulum (ER). Both constructs demonstrate formation of elongated Myc positive & ER negative structures (absence of yellow signal in the merged panels, (c, f, i)) characteristic of pseudo-WPBs (examples are indicated in (a, d), & (g) by arrowheads). Scale bars, 2.5 μm. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/23049555), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
Calnexin Antibody - BSA Free

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974] -

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974] - Zebrafish Vwf forms pseudo-Weibel-Palade bodies (pseudo-WPBs) in mammalian cell culture. pVWF/Myc-HIS (human VWF, (a–c)) or pzVwf/Myc-HIS (zebrafish Vwf, (d–i)) plasmids were transfected into HEK293T cells. Anti-Myc antibody conjugated to Alexa Fluor 488 (green channel, (a, d, g)) was used for detection & anti-calnexin antibody conjugated to Alexa Fluor 594 (red channel, (b, e, h)) labeled endoplasmic reticulum (ER). Both constructs demonstrate formation of elongated Myc positive & ER negative structures (absence of yellow signal in the merged panels, (c, f, i)) characteristic of pseudo-WPBs (examples are indicated in (a, d), & (g) by arrowheads). Scale bars, 2.5 μm. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/23049555), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
Calnexin Antibody - BSA Free

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974] -

Immunocytochemistry/ Immunofluorescence: Calnexin Antibody - BSA Free [NB100-1974] - Zebrafish Vwf forms pseudo-Weibel-Palade bodies (pseudo-WPBs) in mammalian cell culture. pVWF/Myc-HIS (human VWF, (a–c)) or pzVwf/Myc-HIS (zebrafish Vwf, (d–i)) plasmids were transfected into HEK293T cells. Anti-Myc antibody conjugated to Alexa Fluor 488 (green channel, (a, d, g)) was used for detection & anti-calnexin antibody conjugated to Alexa Fluor 594 (red channel, (b, e, h)) labeled endoplasmic reticulum (ER). Both constructs demonstrate formation of elongated Myc positive & ER negative structures (absence of yellow signal in the merged panels, (c, f, i)) characteristic of pseudo-WPBs (examples are indicated in (a, d), & (g) by arrowheads). Scale bars, 2.5 μm. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/23049555), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
Calnexin Antibody - BSA Free

Western Blot: Calnexin Antibody - BSA Free [NB100-1974] -

Western Blot: Calnexin Antibody - BSA Free [NB100-1974] - Liver-specific disruption of Chrebp in mice. A: Quantitative real-time PCR analysis of ChREBP mRNA. Total RNA was isolated from various tissues of control & liver-specific Chrebp knockout (L-Chrebp−/−) mice & subjected to real-time PCR analysis with 36B4 as the invariant control. Each value represents the mean ± SEM of four mice relative to that of controls, which was arbitrarily defined as 1.0. #P < 0.01 denotes the level of statistical significance (two-tailed Student’s t-test) between control & L-Chrebp−/− mice. B: Immunoblot analysis of ChREBP in liver lysates of control & L-Chrebp−/− mice. Aliquots (60 μg of protein) of liver whole-cell lysates were subjected to SDS-PAGE & immunoblot analysis with anti-ChREBP & anti-calnexin antibodies. ChREBP delta denotes a truncated aberrant ChREBP protein present only in lysates prepared from L-Chrebp−/− livers. The functional domains of ChREBP, including the glucose-sensing proline-rich bHLH-Zip DNA-binding & ZIP-like domains, are denoted. NLS, nuclear localization sequence. Image collected & cropped by CiteAb from the following publication (https://linkinghub.elsevier.com/retrieve/pii/S0022227520331369), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
Calnexin Antibody - BSA Free

Western Blot: Calnexin Antibody - BSA Free [NB100-1974] -

Western Blot: Calnexin Antibody - BSA Free [NB100-1974] - Immunoblot analysis of livers from control & L-Chrebp−/− mice subjected to fasting & refeeding with a high-sucrose diet. Littermate control & L-Chrebp−/− mice (same as those described in supplemental Tables S2A & S2B) were subjected to fasting & refeeding. The nonfasted (N) groups were fed chow diet ad libitum. The fasted (F) group was fasted 12 h, & the refed (R) group was fasted for 12 h & then refed with 60% (w/w) high-sucrose diet for 12 h prior to study. Liver whole-cell lysates & membrane fractions were prepared individually, & equal amounts of protein from each mouse of the same group (four per group) were pooled. Aliquots (40 μg for whole-cell lysates & 30 μg for membrane fractions) of the pooled protein were subjected to SDS-PAGE & immunoblot analysis. Immunoblot analysis of Insig-1 & Insig-2 were carried out using membrane fractions. Whole-cell lysates were used to detect other proteins. The precursor & nuclear forms of SREBPs are denoted as P & N, respectively. Calnexin was used as loading control. Image collected & cropped by CiteAb from the following publication (https://linkinghub.elsevier.com/retrieve/pii/S0022227520331369), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Applications for Calnexin Antibody - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:100

Immunohistochemistry

1:100

Immunohistochemistry-Paraffin

1:100

Immunoprecipitation

1:100

Simple Western

1:50

Western Blot

1:1000
Application Notes
In ICC/IF, endoplasmic reticulum staining was observed in HeLa cells. In Western Blot, a band is seen at ~ 90 kDa representing Calnexin. In IHC-P, staining was observed in the endoplasmic reticulum of mouse bladder tissue. Prior to immunostaining paraffin tissues, antigen retrieval with sodium citrate buffer (pH 6.0) is recommended. In Simple Western only 10 - 15 uL of the recommended dilution is used per data point.
See Simple Western Antibody Database for Simple Western validation: Tested in HeLa lysate 0.1 mg/mL, separated by Size, antibody dilution of 1:50, apparent MW was 117 kDa. Separated by Size-Wes, Sally Sue/Peggy Sue.

Reviewed Applications

Read 1 review rated 4 using NB100-1974 in the following applications:

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Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: Calnexin

Calnexin is a calcium-binding protein which interacts with newly synthesized glycoproteins in ER for facilitating protein assembly and/or the retention within ER of unassembled protein subunits as well as incorrectly folded proteins thereby playing a key role in ER's quality control apparatus. The specificity of calnexin for glycoproteins is defined by a lectin site, which binds an early oligosaccharide intermediate on the folding glycoprotein. When associated with partial T-cell antigen receptor complexes which escape immature thymocytes's ER, calnexin regulates thymocyte maturation process and is also implicated in receptor-mediated endocytosis at synapses. MAPK3/ERK1 phosphorylates calnexin at Ser-565 leading to increased calnexin-ribosomal association and DHHC6 mediated calnexin palmitoylation leads to its preferential localization to perinuclear rough ER. Palmitoylation also facilitate calnexin association with RTC/ribosome-translocon complex that is essential to efficient folding of glycosylated proteins.

Alternate Names

calnexin, CNX, IP90FLJ26570, Major histocompatibility complex class I antigen-binding protein p88, P90

Entrez Gene IDs

821 (Human); 29144 (Rat)

Gene Symbol

CANX

UniProt

Additional Calnexin Products

Product Documents for Calnexin Antibody - BSA Free

Certificate of Analysis

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Product Specific Notices for Calnexin Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Citations for Calnexin Antibody - BSA Free

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Protocols

View specific protocols for Calnexin Antibody - BSA Free (NB100-1974):

Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.

Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes (keep slides in the sodium citrate buffer at all times).

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in PBS for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1% BSA in PBS) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul HRP polymer conjugated secondary antibody. Incubate 30 minutes at room temperature.
7. Wash sections three times in wash buffer for 5 minutes each.
8. Add 100-400 ul DAB substrate to each section and monitor staining closely.
9. As soon as the sections develop, immerse slides in deionized water.
10. Counterstain sections in hematoxylin.
11. Wash sections in deionized water two times for 5 minutes each.
12. Dehydrate sections.
13. Mount coverslips.

Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturer's instructions.

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