BMA was mixed with Laemmli sample buffer (1610737 Bio-Rad) and 2-mercaptoethanol; 11 μg (lanes 3 and 9) 18.6 μg (lanes 4 and 10) of protein was loaded into a 4–15% precast gel (4561084 Bio-Rad) and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad) using the Trans Blot Turbo Transfer System (1704150 Bio-Rad). Following blocking with 5% nonfat milk, the membrane was cut in half and incubated with either rabbit anti-c Abl antibody (Ab15130 Abcam) or an isotype control (011-000-003 Jackson Immunoresearch Labs), each diluted to a final concentration of 0.1 μg/mL and incubated overnight at 4°C. After washing with TBS, 0.1% Tween-20, the membrane was incubated with HRP-conjugated anti-rabbit IgG (7074 Cell Signaling Technology) at 1:5000 diluted in TBS, 0.1%Tween-20/5% milk at room temperature for 2 hrs. The membrane was washed again with TBS/0.1%Tween-20 and a chemiluminescent substrate was added (1705060 Bio-Rad) and exposed to x-ray film for various times prior to developing.
Ab15130
Manufactured by Abcam
Sourced in United States
Ab15130 is a primary antibody product from Abcam. It is a polyclonal antibody raised in rabbit and targets the Leucine-rich repeat-containing protein 8A (LRRC8A) protein. The product is suitable for use in applications such as Western Blotting, Immunohistochemistry, and Immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab39012, by Abcam (1 mentions)
Ab52915, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Imagequant las 4000 analyzer, by GE Healthcare (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Ab6728, by Abcam (1 mentions)
Las 4000, by Cytiva (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Vt1000 s vibrating blade microtome, by Leica (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
4 protocols using ab15130
1
Western Blot Analysis of c-Abl Protein
2
Western Blot Analysis of Protein Expression
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Total protein was extracted from tissues or cells, followed by protein concentration assessment by a BCA kit (P0012S, Beyotime, Shanghai, China). Proteins (50 μg) were dissolved in 2× sodium dodecyl sulfate (SDS) loading buffer, then boiled (100°C for 5 min), separated by SDS-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes. Subsequent to blocking in 5% skim milk powder, diluted primary antibodies of ABL1 (ab15130, 1:1,000; Abcam, Cambridge, MA, USA), MMP-3 (ab52915, 1:1,000; Abcam), MMP-13 (ab39012, 1:3,000; Abcam), cleaved-caspase3 (ab32042, 1:500; Abcam), and GAPDH (ab181602, 1:10,000; Abcam) were added for incubation with the membranes at 4°C overnight, prior to incubation with horseradish peroxidase-conjugated secondary antibody goat anti-rabbit immunoglobulin G (IgG) (ab205718, 1:5,000; Abcam) for 2 h after the membrane was washed. Visualization of the protein bands was actualized using electrochemical luminescence, and scanning analysis was performed on a gel imager system. Thereafter, the gray level was determined by Image J software utilizing GAPDH as the internal control.
3
Quantification of Phosphorylated c-Abl in Cells
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After treating LFs with 25 µg mL-1 GNP-HCIm and 10 µM Imatinib for 24 h, cells were lysed. Proteins (10 µg) were loaded onto SDS-PAGE and transferred onto a PVDF membrane using a Trans Blot turbo system (Bio-Rad). After 1 h incubation in 5% Bovine Serum Albumin (BSA) diluted in PBS and three washes with PBS containing 0.1% Tween 20 (TBST), the membrane was incubated overnight with anti-c-Abl (phospho Y412) antibody (ab4717, Abcam) at a 1:1000 dilution in 1% BSA. After three washes with TBST (10 mL), membranes were incubated with Rabbit anti-Mouse IgG H&L (HRP) (ab6728, Abcam) secondary antibody at a 1:2000 dilution in 1% BSA in TBST, for 1 h at room temperature. Clarity Western ECL (Bio-Rad) solution was used according to the provided protocol. The same procedure was applied to identify c-Abl by anti-c-Abl antibody (ab15130, Abcam) at a 1:100 dilution and β-actin (15G5A11/E2, Cat #MA1-140, Thermo Fisher Scientific) at a 1:5000 dilution. Also in this case we used as secondary antibody Rabbit anti-Mouse IgG H&L (HRP) (ab6728, Abcam). All immunoblots were acquired with the ImageQuant LAS 4000 analyzer (GE Healthcare).
4
Immunohistochemistry of c-Abl in Tissue Sections
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Approximately 100 adult males or adult females were fixed in 4% paraformaldehyde (15710 Electron Microscopy Sciences, 16% PFA diluted with 1X PBS) at 4°C overnight, then placed in 2% agarose in DMEM (1054 Gibco). Sections 200 μm thick were sliced with a Leica VT1000 S Vibrating Blade Microtome (Leica Microsystems ) at speed 5, in ice-cold phosphate buffered saline (PBS), and placed in 1% or 2% bovine serum albumin (BSA) in PBS. Sections were permeabilized with 2.5% BSA, 1% Triton-X100 in PBS by agitating slowly at 4°C for 1 hour. Blocking was performed with 1% BSA, 10% goat serum and 0.1% Tween-20 in PBS agitating slowly for 1 hour at 4°C. Sections were washed with 1% BSA/PBS. Polyclonal rabbit antibody raised against human c-Abl (Ab15130 Abcam) or isotype control rabbit IgG (011-000-003 Jackson Immunoresearch Labs) at a concentration 0.05ug/mL was added to 1% BSA, 1% Triton-X100 in PBS and agitated slowly at 4°C for 72–96 hours. Sections were washed twice with 1% BSA/PBS, then counterstained with Alexa Fluor 594 goat anti-rabbit IgG (A31632 Life Technologies) at 1:2000 with 1% Triton-X100, 1%BSA and incubated overnight at 4°C agitating. Sections were then washed with 1%BSA/1% Triton/PBS for 16–24 hours agitating at 4°C. DAPI (R37606 Molecular probes) was then added. The experiment was repeated 6 times.
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