Product nameEndoplasmic Reticulum Fraction Western Blot Cocktail
Sample typeTissue Extracts, Cell Lysate, Tissue Homogenate, Nuclear Extracts
Species reactivityReacts with: Mouse, Rat, Human
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.
Tested applicationsSuitable for: WBmore details
Storage instructionsStore 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
- Pathways and Processes
- Metabolic signaling pathways
- Energy transfer pathways
- Energy Metabolism
Cellular localizationGRP78 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.
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.
|WB||Use at an assay dependent concentration.
Primary antibody cocktail = 1/250 dilution
Secondary antibody cocktail = 1/2500 dilution
Developed 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
Lane 1: Marker
Lanes 2-5: Hela Whole Cell Lysate – 20 µg
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
HeLa 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.
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
Developed 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.
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
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.
ab139415 has not yet been referenced specifically in any publications.