Endoplasmic Reticulum Fraction Western Blot Cocktail (ab139415)
Key features and details
- Sample type: Cell Lysate, Nuclear Extracts, Tissue Extracts, Tissue Homogenate
Overview
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Product name
Endoplasmic Reticulum Fraction Western Blot Cocktail -
Sample type
Tissue Extracts, Cell Lysate, Tissue Homogenate, Nuclear Extracts -
Species reactivity
Reacts with: Mouse, Rat, Human -
Product overview
ab139415 contains 3 mAbs each targeting a specific organelle marker. The presence of endoplasmic reticulum is detected by GRP78; cytosol by Anti-GAPDH; and nucleus by Anti-Histone H3 (di methyl K9). This cocktail is suitable for determining the purity of organelle isolates prior to further characterization.
This product is particularly valuable to researchers working in organelle proteomics. Mass spectrometry is frequently used in this field to determine the protein content of targeted organelle isolates. These isolates are obtained using differential centrifugation, density gradient fractionation, biochemical enrichment, or affinity purification. Unfortunately, the various methods of purification available for organelle isolation are imperfect and leave behind contaminants from undesired regions of the cell. These contaminants are inevitable, but being aware of which contaminants are present is crucial for analysis of mass spectrometry results. The high sensitivity and species cross reactivity of the antibodies in this cocktail will quickly and easily reveal impurities caused by imperfect sample preparation.
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Tested applications
Suitable for: WBmore details
Properties
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Storage instructions
Store at -20°C. Please refer to protocols. -
Components 200 µl 2500X HRP Conjugated Secondary Antibody Cocktail 1 x 50µl 250X Endoplasmic Reticulum Fraction Western Blot Cocktail 1 x 200µl -
Research areas
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Cellular localization
GRP78 BiP: Endoplasmic reticulum lumen. Melanosome. Cytoplasm. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. GAPDH: Cytoplasm > cytosol. Nucleus. Cytoplasm > perinuclear region. Membrane. Translocates to the nucleus following S-nitrosylation and interaction with SIAH1, which contains a nuclear localization signal (By similarity). Postnuclear and Perinuclear regions. Histone H3: Nucleus. Chromosome. -
Database links
- Entrez Gene: 2597 Human
- Entrez Gene: 3309 Human
- Entrez Gene: 8350 Human
- Entrez Gene: 8351 Human
- Entrez Gene: 8352 Human
- Entrez Gene: 8353 Human
- Entrez Gene: 8354 Human
- Entrez Gene: 8355 Human
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Associated products
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab139415 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| WB |
Use at an assay dependent concentration.
Primary antibody cocktail = 1/250 dilution Secondary antibody cocktail = 1/2500 dilution |
| Notes |
|---|
|
WB
Use at an assay dependent concentration. Primary antibody cocktail = 1/250 dilution Secondary antibody cocktail = 1/2500 dilution |
Images
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Western Blot for Endoplasmic Reticulum Membrane Antibody Cocktail – Component SeparationDeveloped using the ECL technique; Performed under reducing conditions; Exposure time: 5 mins. All blocking and antibody incubation steps were performed in 5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20
Samples
Lane 1: Marker
Lanes 2-5: Hela Whole Cell Lysate – 20 µg
Primary antibody:
Lane 1: None
Lane 2: Anti-GRP78 antibody – Endoplasmic Reticulum Marker
Lane 3: Anti- GAPDH antibody – Cytosolic Marker
Lane 4: Anti- Histone H3 (di methyl K9) antibody – Nuclear Marker
Lane 5: Assembled Endoplasmic Reticulum Antibody Cocktail
Secondary:HRP conjugated secondary antibody cocktail at 1/2500
Predicted GRP78 band size: 78 kDa
Observed band size: 78 kDa
Predicted GAPDH band size: 37 kDa
Observed band size: 37 kDa
Predicted Histone H3 (di methyl K9) band size: 17 kDa
Observed band size: 17 kDa -
Western Blot for Endoplasmic Reticulum Membrane Antibody Cocktail – Cell FractionationHeLa cell lysates were prepared using the Membrane Fractionation Kit (ab139409), blots were developed using the ECL technique, performed under reducing conditions and exposed for 5 minutes. A 4-15% gradient acrylamide gel was used and blocking and antibody incubation steps were performed in 5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20.
Sample lanes:
Lane 1 – Marker
Lane 2 - HeLa whole cell lysate - 20 µL
Lane 3 - HeLa cytosolic fraction lysate - 20 µL
Lane 4 - HeLa membrane fraction lysate - 20 µL
Lane 5 - HeLa nuclear fraction lysate - 20 µL
Primary antibody: ER Fraction WB cocktail at 1/250 dilution
Secondary antibody: HRP conjugated secondary cocktail at 1/2500.
Predicted/observed band sizes:
GRP78: 78 kDa/78 kDa
GAPDH: 37 kDa/37 kDa
Histone H3 (di methyl K9): 17 kDa/17 kDa -
Western Blot for Endoplasmic Reticulum Membrane Antibody Cocktail – Cross ReactivityDeveloped using the ECL technique, performed under reducing conditions and exposed for 3 minutes. All blocking and antibody incubation steps performed in 5% milk, 20 mM Tris-HCl, 0.1% TWEEN-20.
Samples:
1: Marker
2: Human heart homogenate lysate - 20 µg
3: HeLa cell lysate - 20 µg
4: Mouse heart homogenate lysate - 20 µg
5: NIH3T3 cell lysate - 20 µg
6: Rat heart homogenate lysate - 20 µg
7: H9C2 cell lysate - 20 µg
Primary antibody:ER Fraction WB cocktail at 1/250.
Secondary antibody: HRP conjugated secondary antibody cocktail at 1/2500.
Predicted/Observed band sizes:
GRP78: 78 kDa/75 kDa
GAPDH: 37 kDa/37 kDa
Histone H3 (dimethyl K9): 17 kDa/17 kDa
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (2)
ab139415 has been referenced in 2 publications.
- Pérez-Rodriguez S et al. Enrichment of microsomes from Chinese hamster ovary cells by subcellular fractionation for its use in proteomic analysis. PLoS One 15:e0237930 (2020). PubMed: 32841274
- Micakovic T et al. Isolation of Pure Mitochondria from Rat Kidneys and Western Blot of Mitochondrial Respiratory Chain Complexes. Bio Protoc 9:e3379 (2019). PubMed: 33654875