Performed under reducing conditions. All blocking and antibody incubation steps were done in 5% milk in TBST. Developed using the ECL technique. Exposure time: 5 mins.

Sample 1: Marker
Samples 2-5: Hela Cell Lysate – 20 µg

Primary:
Lane 1: none
Lane 2: Anti- Sodium Potassium ATPase antibody – Plasma Membrane Marker
Lane 3: Anti-GRP78 antibody – Endoplasmic Reticulum Marker
Lane 4: Anti- ATP5a antibody – Mitochondrial Membrane Marker
Lane 5: Anti- GAPDH antibody – Cytosolic Marker
Lane 6: Anti- Histone H3 (di methyl K9) antibody – Nuclear Marker
Lane 7: Assembled Membrane Antibody Cocktail

Secondary:HRP conjugated secondary antibody cocktail

Predicted/observed band sizes:
Sodium Potassium ATPase = 112 kDa/100 kDa
GRP78 band size: 78 kDa / 75 kDa
ATP5A band size: 60 kDa / 60 kDa
GAPDH band size: 37 kDa / 38 kDa
Histone H3 (di methyl K9) band size: 17 kDa / 17 kDa

Developed by IR scanning. HeLa cell lysate prepared using Kit ab139409. Exposure time: 5 mins . Samples run on a 4-20% gradient acrylamide gel. All blocking and antibody incubation steps were done in 5% milk, 20mM Tris-HCl, 0.1% TWEEN-20
Lane 1: Marker
Lane 2 : HeLa Whole Cell Lysate
Lane 3 : HeLa Cytosolic Fraction Lysate
Lane 4 : HeLa Membrane Fraction Lysate
Lane 5 : HeLa Nuclear Fraction Lysate
Primary antibody: Membrane Fraction Western Blot cocktail
Secondary: IR800 GAM (1/10000) and IR690 GAR (1/10000)
Predicted/Observed band sizes:
Sodium Potassium ATPase: 112 kDa/100 kDa
GRP78: 78 kDa/75 kDa
ATP5A: 60 kDa/60 kDa
GAPDH: 37 kDa/37kDa
Histone H3 (di methyl K9): 17 kDa/17kDa
Notes: Percentage of antibody signal (Y axis) represents the signal present in individual fractions as a proportion of the sum of all three fractions (cytoplasmic, membrane and nuclear)

Developed using ECL technique under reducing conditions. HeLa lysate prepared using the Membrane Fractionation Kit (ab139409). Exposure time: 5 mins. on a 4-20% gradient acrylamide gel, blocking and antibody incubation steps done in 5% milk, 20mM Tris-HCl, 0.1% TWEEN-20
Lane 1: Marker
Lane 2 : HeLa Whole Cell Lysate
Lane 3 : HeLa Cytosolic Fraction Lysate
Lane 4 : HeLa Membrane Fraction Lysate
Lane 5 : HeLa Nuclear Fraction Lysate
Primary antibody: Membrane Fraction Western Blot cocktail
Secondary: HRP conjugated secondary antibody cocktail
Predicted/Observed band sizes:
Sodium Potassium ATPase: 112 kDa/100 kDa
GRP78: 78 kDa/75 kDa
ATP5A: 60 kDa/60 kDa
GAPDH: 37 kDa/37kDa
Histone H3 (di methyl K9): 17 kDa/17kDa
Notes: Percentage of antibody signal represents signal present in individual fractions as a proportion of the sum of all three fractions (cytoplasmic, membrane and nuclear)

Samples run on a 4-20% gradient acrylamide gel. Performed under reducing conditions. All blocking and antibody incubation steps were done in 5% milk, 20mM Tris-HCl, 0.1% TWEEN-20. Developed using the ECL technique. Exposure time: 5 mins.

Lane 1: Marker
Lane 2: Human heart homogenate Lysate – 20 µg
Lane 3: Hela Cell Lysate – 20 µg
Lane 4: Mouse heart homogenate Lysate - 20 µg
Lane 5: NIH3T3 Cell Lysate – 20 µg
Lane 6: Rat heart homogenate Lysate – 20 µg
Lane 7: H9C2 Cell Lysate – 20 µg

Primary antibody: Membrane Fraction Western blot cocktail at 1/250 dilution

Secondary antibody: HRP conjugated secondary antibody cocktail at 1/2500 dilution

Predicted/observed band sizes:
Sodium Potassium ATPase = 112 kDa/100 kDa
GRP78 band size: 78 kDa / 75 kDa
ATP5A band size: 60 kDa / 60 kDa
GAPDH band size: 37 kDa / 38 kDa
Histone H3 (di methyl K9) band size: 17 kDa / 17 kDa