ATP7b Antibody - BSA Free

Novus Biologicals | Catalog # NB100-360

Novus Biologicals
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Key Product Details

Species Reactivity

Validated:

Human, Mouse, Rat

Cited:

Human, Mouse, Rat

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Immunoblotting, Immunocytochemistry/ Immunofluorescence, Immunoprecipitation

Cited:

Immunohistochemistry-Paraffin, Western Blot, Immunoblotting, Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunoprecipitation, IF/IHC

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all ATP7b Primary Antibodies »

Product Specifications

Immunogen

A synthetic peptide made to an internal sequence near the N-terminus of human ATP7b. [UniProt# P35670]

Localization

Golgi apparatus; Predominantly found in trans-Golgi network membrane; multi-pass membrane protein (By similarity). Isoform 2: Cytoplasm. The cleaved form WND/140 kDa is located in mitochondria.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

165 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 ATP7b Antibody - BSA Free

Western Blot: ATP7b AntibodyBSA Free [NB100-360]

Western Blot: ATP7b AntibodyBSA Free [NB100-360]

Western Blot: ATP7b Antibody [NB100-360] - ATP7b detected in 20 ug of mouse brain membrane fraction.
Immunocytochemistry/ Immunofluorescence: ATP7b Antibody - BSA Free [NB100-360]

Immunocytochemistry/ Immunofluorescence: ATP7b Antibody - BSA Free [NB100-360]

Immunocytochemistry/Immunofluorescence: ATP7b Antibody [NB100-360] - HeLa cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton X-100. The cells were incubated with anti-ATP7b [NB100-360] at a 1:100 dilution overnight at 4C and detected with an anti-rabbit DyLight 488 (Green) at a 1:500 dilution. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse DyLight 550 (Red) at 1:500. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Immunohistochemistry: ATP7b Antibody - BSA Free [NB100-360]

Immunohistochemistry: ATP7b Antibody - BSA Free [NB100-360]

Immunohistochemistry: ATP7b Antibody [NB100-360] - Analysis of ATP7b in mouse liver using DAB with hematoxylin counterstain.
ATP7b Antibody - BSA Free

Western Blot: ATP7b Antibody - BSA Free [NB100-360] -

Copper contributes to the production of MB in Wilson disease through inhibiting autophagy. (A) Differential detergent fractionation was performed on WT HepG2 cells or GFP-Ctr1 HepG2 after treatment with 1 mM of copper ions for 6 h. Western blotting was carried out to detect the protein levels. (B) Colocalization analysis of the markers of MB such p62, K8/K18, and Ub before and after treatment of copper ions. The arrows mean colocalization. (C) Differential detergent fractionation was performed on WT HepaRG cells or ATP7B knockdown HepRG cells treated with 1 mM of copper ions for 6 h and western blotting was carried out to detect the protein levels. (D) Differential detergent fractionation followed by western blotting was performed on HepaRG cells treated with 1 mM of copper ions in the presence or absence of 40 μM of CQ for 6 h. (E) Differential detergent fractionation was performed on ATP7B knockdown HepRG cells treated with 1 mM of copper ions for 6 h and western blotting was carried out to detect the protein levels. (F) Densitometry analysis of protein bands of (E) was performed. The ratio of K8/K18 and LC3B in soluble fraction, p62 and ubiquitin in insoluble fraction was calculated. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35349929), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Applications for ATP7b Antibody - BSA Free

Application
Recommended Usage

Immunoblotting

reported in scientific literature (PMID 29250175)

Immunocytochemistry/ Immunofluorescence

2 - 10 ug/mL

Immunohistochemistry

1:100 - 1:500

Immunohistochemistry-Paraffin

1:100 - 1:500

Western Blot

1:1000
Application Notes
In WB this antibody recognizes a band at 165 kDa, representing ATP7b and also recognizes a faint non-specific band at ~195 kDa. In IHC-P, cytoplasmic and golgi staining was observed in mouse liver tissue (1:100 dilution) and human ovarian carcinoma cells (1:500 dilution). However, please note we also tested this antibody with IHC-P in mouse intestines and kidney and observed unexpected strong nuclear signal with lighter cytoplasmic staining. Prior to immunostaining paraffin tissues, antigen retrieval with sodium citrate buffer (pH 6.0) is recommended. 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.

Reviewed Applications

Read 1 review rated 4 using NB100-360 in the following applications:

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. Do not freeze.

Background: ATP7b

ATP7b (copper-transporting P-type adenosine triphosphatase) is also called copper-transporting ATPase 2 or Wilson disease-associated protein and is involved in copper export out of the cells, such as the efflux of hepatic copper into the bile. ATP7b is a monomeric protein that interacts with COMMD1/MURR1, DCTN4 (in copper-dependent manner) and ATOX1. Predominantly localized in the trans-Golgi network (TGN), ATP7b is most abundantly expressed in liver, kidney and the brain tissues (isoform 2 is expressed in cytoplasm of brain cells but not in liver). Isoform 1 is proteolytically cleaved at N-terminus to produce the WND/140 kD form which is found in mitochondrions of hepatocytes as well as other tissue/cell types. Defects in ATP7B have been linked to Wilson disease (WD), an autosomal recessive disorder of copper metabolism, wherein, copper cannot be incorporated into hepatic ceruloplasmin and cannot be excreted from the liver into the bile.

Alternate Names

ATPase, Cu(2+)- transporting, beta polypeptide, ATPase, Cu++ transporting, beta polypeptide, Copper pump 2, copper-transporting ATPase 2, EC 3.6.3, EC 3.6.3.4, PWD, WC1, WD, Wilson disease-associated protein, WNDATPase, Cu++ transporting, beta polypeptide (Wilson disease)

Gene Symbol

ATP7B

Additional ATP7b Products

Product Documents for ATP7b Antibody - BSA Free

Certificate of Analysis

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Product Specific Notices for ATP7b 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 ATP7b Antibody - BSA Free

Customer Reviews for ATP7b Antibody - BSA Free (1)

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  • Name: Anonymous
    Application: Western Blot
    Sample Tested: Whole cell lysates for ovarian cancer cell lines A2780 and its cisplatin resistant variant A2780-CP20.
    Species: Human
    Verified Customer | Posted 09/07/2015
    Western Blot for ATP7b
    ATP7b Antibody - BSA Free NB100-360

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Protocols

View specific protocols for ATP7b Antibody - BSA Free (NB100-360):


Immunocytochemistry Protocol

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

1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three times for 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to wash solution at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.


Immunohistochemistry Procedure

Cell Preparation (At least 108 cells were used per block)

1. Harvesting cells:

A. Trypsinization
B. 15 minute centrifugation at 2,500 RPM
C. PBS rinse
D. 15 minute centrifugation at 2,500 RPM

2. Suspend cells in 10 ml of 10% formaldehyde in PBS, overnight @ RT.
3. Centrifuge cells at 2,500 RPM for 10 minutes.
4. Resuspend cells in 10 ml of 70% ethanol.
5. Centrifuge cells at 2,500 RPM and taken into 70% ethanol.


Cell Staining

1. Ribbon Thickness: 5 um
2. Deparaffination Agent: Xylin
3. Hydration: Ethanol in PBS
4. Antigen Retrieval: 10 minute microwave retrieval in citrate buffer; 20 minute cooling
5. Blocking:

A. endogeneous peroxidase: 0.3% H2O2 in PBS for 10 minutes
B. endogeneous protein: 1% BSA for 20 minutes

6. Primary antibody, polyclonal anti-ATP7b (NB 100-360): 1:500, overnight @ 4 degrees Celcius
7. Secondary antibody, anti-rabbit (HRP): (dilute per manufacturer recommendation), 30 minutes @ RT
8. Wash 3x 15 minutes
9. Chromogen: AEC
10. Counterstain: Mayers hematoxylin

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.


Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 40 ug of total protein per lane.
2. Transfer proteins to membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain according to standard Ponceau S procedure (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot.
5. Block the membrane using standard blocking buffer for at least 1 hour.
6. Wash the membrane in wash buffer three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate 1 hour at room temperature.
8. Wash the membrane in wash buffer three times for 10 minutes each.
9. Apply the diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer 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 manufacturers instructions.
Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.

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FAQs for ATP7b Antibody - BSA Free

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  • Q: Can you disclose any further detail related to the immunogen sequence used to generate NB100-360?

    A: A more specific range for the immunogen of our NB100-360 is aa300-350 of the human ATP7b protein 

  • Q: Which isoform of the protein does NB100-360 recognize?

    A: The immunogen for this product is to the N-terminal, so it will recognize all 4 known isoforms of ATP7b.

  • Q: Would you be able to provide any additional information about the sequence or location of the immunogenic peptide?

    A: I cannot give the exact immunogen sequence as it is considered to be propriety information; however, I am able to provide a range. The immunogen of NB100-360 lies within aa 300-350 of the human ATP7b protein.

  • Q: Can you disclose any further detail related to the immunogen sequence used to generate NB100-360?

    A: A more specific range for the immunogen of our NB100-360 is aa300-350 of the human ATP7b protein 

  • Q: Which isoform of the protein does NB100-360 recognize?

    A: The immunogen for this product is to the N-terminal, so it will recognize all 4 known isoforms of ATP7b.

  • Q: Would you be able to provide any additional information about the sequence or location of the immunogenic peptide?

    A: I cannot give the exact immunogen sequence as it is considered to be propriety information; however, I am able to provide a range. The immunogen of NB100-360 lies within aa 300-350 of the human ATP7b protein.

  • Q: Can you disclose any further detail related to the immunogen sequence used to generate NB100-360?

    A: A more specific range for the immunogen of our NB100-360 is aa300-350 of the human ATP7b protein 

  • Q: Which isoform of the protein does NB100-360 recognize?

    A: The immunogen for this product is to the N-terminal, so it will recognize all 4 known isoforms of ATP7b.

  • Q: Would you be able to provide any additional information about the sequence or location of the immunogenic peptide?

    A: I cannot give the exact immunogen sequence as it is considered to be propriety information; however, I am able to provide a range. The immunogen of NB100-360 lies within aa 300-350 of the human ATP7b protein.

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