Anti-CPT1A antibody [8F6AE9]

• WB

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Western blot - Anti-CPT1A antibody [8F6AE9] (AB128568)

Predicted partial recombinant protein band size : 32 kDa
Observed band size : 32 kDa

All lanes:

Western blot - Anti-CPT1A antibody [8F6AE9] (ab128568) at 1 µg/mL

Lane 1:

Marker

Lane 2:

Partial Human Recombinant CPT1A protein (<a href='/en-us/products/unavailable/recombinant-human-cpt1a-protein-ab128569'>ab128569</a>) at 0.1 µg

Lane 3:

Full Length Human Recombinant OTC protein at 0.1 µg

Lane 4:

HepG2 (Human hepatocellular carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 5:

H9C2 (Rat cardiomyoblast cell line) Whole Cell Lysate at 20 µg

Lane 6:

H4IIE ( Rat hepatoma cell line) Whole Cell Lysate at 20 µg

Lane 7:

RHM (Rat heart mitochondrial homogenate) at 20 µg

Lane 8:

HLH (Human liver homogenate) at 20 µg

Lane 9:

RLH (Rat liver homogenate) at 20 µg

Lane 10:

MLH (Mouse liver homogenate) at 20 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG – H&L – Pre-Adsorbed (HRP) at 1/10000 dilution

Predicted band size: 88 kDa

Observed band size: 88 kDa

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Exposure time: 1min

• Flow Cyt

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Flow Cytometry - Anti-CPT1A antibody [8F6AE9] (AB128568)

Overlay histogram showing HeLa cells stained with ab128568 (red line) or no primary antibody (black line). The cells were fixed with 4% paraformaldehyde (15 min) and then permeabilized with 0.1% Triton X-100 in PBS, 3% BSA for 10 min. The cells were then incubated in 3% BSA in PBS for 10 minutes to block non-specific protein-protein interactions followed by the antibody (ab128568, 1µg/mL) for 60 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96871) at 1/1000 dilution for 30 min.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CPT1A antibody [8F6AE9] (AB128568)

IHC image of CPT1A staining in human normal kidney formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab128568, 0.5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CPT1A antibody [8F6AE9] (AB128568)

Immunohistochemical analysis of formalin fixed paraffin embedded human kidney labelling CPT1A with ab128568 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC2) 100°C, pH8.5 for 32mins.

ab128568 anti-CPT1A [8F6AE9] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• In-Cell ELISA

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In-Cell ELISA - Anti-CPT1A antibody [8F6AE9] (AB128568)

In-Cell ELISA for Anti-CPT1A antibody (ab128568) stained HeLa cells (human)

Seeding : HeLa cells seeded in a 1 : 2 dilution series starting at 60,000 cells/well across Row A, 30,000 cells/well across Row B, etc. Row H contains no cells.

Table : Example In-Cell ELISA Average Data from shown plate.

Fixation : 4% paraformaldehyde PBS fixed for 15 minutes

Permeabilization : 0.3% Triton X-100 PBS for 30 minutes at room temperature while shaking

Blocking : 2x Sigma Block 0.3% Triton X-100 H2O for 2 hours at room temperature while shaking

Primary antibodies : All primaries diluted in 1x Sigma Block with 0.3% Triton X-100 incubated overnight at 4 °C.

- Columns 1-3 : Anti-CPT1A antibody (ab128568) 10 ug/mL

- Columns 4-6 : Anti-CPT1A antibody (ab128568) 1 ug/mL

- Columns 7-9 : Anti-CPT1A antibody (ab128568) 0.1 ug/mL

- Columns 10-12 : No Primary

Washing : Briefly 4x with 0.3% TWEEN-20 PBS

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CPT1A antibody [8F6AE9] (AB128568)

Immunofluorescent staining of CPT1A in H9C2 cells (rat) using ab128568.

Fixation : 4% paraformaldehyde PBS fixed for 20 minutes
Permebilization : 0.1% Triton X-100 PBS for 30 minutes at room temperature while rocking
Blocking : 2x Sigma Block 0.1% Triton X-100 H2O for 2 hours at room temperature while rocking
Primary antibodies : Anti-CPT1A antibody (ab128568) 0.5 ug/mL 1x Sigma Block with 0.1% Triton X-100 incubated overnight at 4 °C.
Washing : 3x 1% NGS 10 minutes/wash.
Secondary antibodies : Alexa 488 GAM 1 : 1000 diluted in 1% NGS with 0.1% Triton X-100 PBS incubated for 2 hours at room temperature while rocking.
Washing : 3x 1% NGS 10 minutes/wash.
DAPI : 20 ng/mL in 1% NGS, 0.1% Triton X-100 PBS.
Washing : 1x 1% NGS 10 minutes/wash.

• WB

Lab

Western blot - Anti-CPT1A antibody [8F6AE9] (AB128568)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : CPT1A knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : MCF-7 cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab128568 observed at 88 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab128568 specifically detected the expected band for CPT1A in wild-type HAP1 cells. No band was observed in CPT1A knockout cells. Wild-type and CPT1A knockout samples were subjected to SDS-PAGE. ab128568 and ab181602 (loading control to GAPDH) were diluted at 1 μg/mL and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CPT1A antibody [8F6AE9] (ab128568)

Predicted band size: 88 kDa

false

• WB

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Western blot - Anti-CPT1A antibody [8F6AE9] (AB128568)

All lanes:

Western blot - Anti-CPT1A antibody [8F6AE9] (ab128568) at 1 µg/mL

Lane 1:

Marker

Lane 2:

HHH (Human heart homogenate) RIPA Extract Immunoprecipitated with no primary antibody

Lane 3:

HHH (Human heart homogenate) RIPA Extract Immunoprecipitated with Anti-CPT1A antibody 8F6AE9 (ab128568)

Lane 4:

HHH (Human heart homogenate) RIPA Extract 20 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG – H&L – Pre-Adsorbed (HRP) at 1:10000

Predicted band size: 88 kDa

true

Exposure time: 1min