Anti-ABCA1 antibody [EPR27494-51]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ABCA1 antibody [EPR27494-51] (AB307534)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeABilized U-87 MG (human glioblastoma-astrocytoma epithelial cell) cells lABelling ABCA1 with ab307534 at 1/500 (1.042 ug/ml) dilution, followed by ab150081 Goat Anti-RABbit IgG H&L (Alexa Fluor? 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing membranous and cytoplasmic staining in U-87 MG cell line.Negative control : 293T (PMID : 11896206).Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor? 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-RABbit IgG H&L (Alexa Fluor? 488) preadsorbed at 1/1000 2ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-ABCA1 antibody [EPR27494-51] (AB307534)

ABCA1 was immunoprecipitated from 0.35 mg U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate 10 ug with ab307534 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307534 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate 10 ug
Lane 2 : ab307534 IP in U-87 MG whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307534 in U-87 MG whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 24 seconds

All lanes:

Immunoprecipitation - Anti-ABCA1 antibody [EPR27494-51] (ab307534) at 1/1000 dilution

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate 10 μg

Lane 2:

U-87 MG whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 260 kDa

false

Exposure time: 24s

• WB

Lab

Western blot - Anti-ABCA1 antibody [EPR27494-51] (AB307534)

Blocking and dilution buffer : 5% NFDM/TBST.

The higher band is probably the glycosylated form (PMID : 16873719).

All lanes:

Western blot - Anti-ABCA1 antibody [EPR27494-51] (ab307534) at 1/1000 dilution

Lane 1:

Untreated THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 2:

THP-1 treated first with 100ng/ml PAM for 72 hours and then replaced with 100ng/ml LPS for 5 hours, 300ng/ml BFA was then added for additional 3 hours whole cell lysate at 20 µg

Lane 3:

Untreated HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HepG2 treated with 20uM T0901317 for 16 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 254 kDa

Observed band size: 254 kDa,400 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-ABCA1 antibody [EPR27494-51] (AB307534)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure : lanes 1-4 : 180 seconds; lanes : 5-8; 10 seconds

The higher band is probably glycosylated form (PMID : 16873719).

Compared with ab307536, ab307534 has higher sensitivity.

We recommend ab307534 as an alternative in testing human lysate, or optimize experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to get visible band.

Lanes 1 - 4:

Western blot - Anti-ABCA1 antibody [EPR27494-57] (<a href='/en-us/products/primary-antibodies/abca1-antibody-epr27494-57-ab307536'>ab307536</a>) at 1/1000 dilution

Lanes 5 - 8:

Western blot - Anti-ABCA1 antibody [EPR27494-51] (ab307534) at 1/1000 dilution

Lane 6:

THP-1 treated first with 100ng/ml PAM for 72 hours and then replaced with 100ng/ml LPS for 5 hours, 300ng/ml BFA was then added for additional 3 hours whole cell lysate at 20 µg

Lane 7:

Untreated HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 1 and 5:

Untreated THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 2:

THP-1 treated first with 100ng/ml PAM for 72 hours and then replaced with 100ng/ml LPS for 5 hours, 300ng/ml BFA was then added for additional 3 hours whole cell lysate at 20 µg

Lane 3:

Untreated HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 4 and 8:

HepG2 treated with 20uM T0901317 for 16 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 254 kDa

Observed band size: 254 kDa,400 kDa

false

• WB

Supplier Data

Western blot - Anti-ABCA1 antibody [EPR27494-51] (AB307534)

Blocking and diluting buffer and concentration : 5% NFDM/TBSTNegative control : 293T(PMID : 11896206), SH-SY5Y(PMID : 23600914). Bands below 260 kDa may due to degradation. Samples are non-boiled as boiling may cause protein aggregates. Exposure time :

All lanes:

Western blot - Anti-ABCA1 antibody [EPR27494-51] (ab307534) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate 40 μg

Lane 2:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate 40 μg

Lane 3:

293T (human embryonic kidney epithelial cell) whole cell lysate 40 μg

Lane 4:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate 40 μg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 260 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-ABCA1 antibody [EPR27494-51] (AB307534)

Blocking and diluting buffer and concentration : 5% NFDM/TBSTSamples are non-boiled as boiling may cause protein aggregates. This antibody does not cross-react with human ABCA3, ABCA4 and ABCA7. Exposure time : 3 seconds

All lanes:

Western blot - Anti-ABCA1 antibody [EPR27494-51] (ab307534) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate 20 μg

Lane 2:

293T cells transfected with an empty vector containi a myc-his tag whole cell lysate 20 μg

Lane 3:

293T cells transfected with a human CA1 expression vector containi a myc-his tag whole cell lysate 10 μg

Lane 4:

293T cells transfected with a human CA3 expression vector containi a myc-his tag whole cell lysate 36 μg

Lane 5:

293T cells transfected with a human CA4 expression vector containi a myc-his tag whole cell lysate 4 μg

Lane 6:

293T cells transfected with a human CA7 expression vector containi a myc-his tag whole cell lysate 4 μg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 260 kDa

false

Exposure time: 3s