PA5-32254

antibody from Invitrogen Antibodies

Targeting: MAP1LC3B ATG8F

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunoprecipitation (Data presented on provider website)

Immunohistochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-32254

Provider

Invitrogen Antibodies

Product name

LC3B Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

Recommended positive controls: NT2D1, PC-3, U87-MG, SK-N-SH, mouse brain, Rat brain, HepG2 (untreated), HepG2 (3 µM Thapsigargin treatment for 12 hr), HepG2 (3 µM Thapsigargin treatment for 16hr), HepG2 (3 µM Thapsigargin treatment for 24hr), Huh7 (un-infected), Huh7 (HCV-infected), HeLa, HeLa (50 µM Chloroquine treatment for 24 hr). Predicted reactivity: Zebrafish (100%), Japanese Medaka (100%), Pig (100%), Rhesus Monkey (100%), Chimpanzee (100%), Bovine (100%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.

Reactivity

Human, Mouse, Rat, Porcine

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

0.34 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of LC3B1+LC3B2 using A) 30 µg HepG2 whole cell lysate (untreated) and B) 30 µg HepG2 whole cell lysate (3 µM Thapsigargin treatment for 12 hr). Samples were loaded onto a 15% SDS-PAGE gel and probed with a LC3B1+LC3B2 polyclonal antibody (Product # PA5-32254) at a dilution of 1:1000. ACTB was used as an internal control at a dilution of 1:20,000, shown at the bottom panel.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Human naïve CD4 T cells were activated with anti-CD3/CD28 beads (1:1) (Product # 11132D) for 3 days. Western blot analysis of LC3-B was performed with whole cell lysate. Proteins were transferred to PVDF and then blocked in blocking buffer (TBST+5% non-fat milk) for one hour at room temperature. LC3-B was detected using a LC3B polyclonal antibody (Product # PA5-32254) at a dilution of 1:1,000 overnight at 4°C on a rocking platform. Goat anti Rabbit HRP-conjugated secondary antibody was diluted at 1:2000 in 1xTBST buffer with 5% non fat milk and incubated for 1 hour at room temperature. Data courtesy of the antibody data exchange program.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot analysis of LC3B was performed by separating 30 µg of whole cell extracts by 15% SDS-PAGE. Proteins were transferred to a membrane and probed with a LC3B Polyclonal Antibody (Product # PA5-32254) at a dilution of 1:1000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

LC3B Polyclonal Antibody detects LC3B protein by western blot analysis. Various whole cell extracts (30 µg) were separated by 15% SDS-PAGE, and the membrane was blotted with LC3B Polyclonal Antibody (Product # PA5-32254) diluted at a dilution of 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot analysis of LC3B was performed by separating 30 µg of untreated (–) and treated (+) HeLa whole cell extracts by 15% SDS-PAGE. Proteins were transferred to a membrane and probed with a LC3B Polyclonal Antibody (Product # PA5-32254) at a dilution of 1:2500.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

LC3B Polyclonal Antibody detects LC3B protein in HCV-infected samples by western blot analysis. A. 20 µg Huh7 whole cell lysate/extract (un-infected). B. 20 µg Huh7 whole cell lysate/extract (HCV-infected). LC3B Polyclonal Antibody (Product # PA5-32254) dilution: 1:1500.The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot analysis of LC3B was performed by separating 30 µg of untreated (–) and treated (+) HepG2 whole cell extracts by 15% SDS-PAGE. Proteins were transferred to a membrane and probed with a LC3B Polyclonal Antibody (Product # PA5-32254) at a dilution of 1:1000.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

LC3B Polyclonal Antibody detects MAP1LC3B protein by western blot analysis. A. 20 µg Huh7 whole cell lysate/extract (untreated). B. 20 µg Huh7 whole cell lysate/extract (3 µM-Thapsigargin treatment for 12hr). LC3B Polyclonal Antibody (Product # PA5-32254) dilution: 1:1500. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

LC3B Polyclonal Antibody detects MAP1LC3B protein by western blot analysis. A. 50 µg mouse brain lysate/extract.15% SDS-PAGE. LC3B Polyclonal Antibody (Product # PA5-32254) dilution: 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

LC3B Polyclonal Antibody detects MAP1LC3B protein by western blot analysis. A. 50 µg Rat brain lysate/extract.15% SDS-PAGE. LC3B Polyclonal Antibody (Product # PA5-32254) dilution: 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-LC3B Mouse Polyclonal Antibody (Product # PA5-32254) and 14-16 kDa bands corresponding to LC3B were observed in cell lines tested upon. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HeLa treated with Chloroquine (50uM for 12 Hours) (Lane 2), HCT116 (Lane 3) and HCT 116 treated with Chloroquine (Lane 4) were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence Goat Anti-Mouse IgG Secondary Antibody, HRP conjugate (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of LC3B1+LC3B2 showing staining in the autophagosome of Hep G2 cells. Hep G2 cells mock (left) and treated with 3µM Thapsigargin for 12 hrs (right) were fixed in ice-cold MeOH for 10 min and permeabilized with ice-cold acetone for 1 min and stained using a LC3B1+LC3B2 polyclonal antibody (Product # PA5-32254) diluted at 1:500. Blue: Hoechst 33342 staining. Scale bar = 10µm.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of LC3B1+LC3B2 showing staining in the autophagosome of HeLa cells. HeLa cells mock (left) and treated with 50µM Chloroquine for 24 hr (right) were fixed in 4% paraformaldehyde at RT for 15 min and stained using a LC3B1+LC3B2 polyclonal antibody (Product # PA5-32254) diluted at 1:1000. Red: Phalloidin, a F-actin marker.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunocytochemistry-Immunofluorescence analysis of LC3B in HeLa cells mock (left) and in HeLa cells treated with 50 µM Chloroquine for 24 hr (right) fixed with 4% paraformaldehyde at RT for 15 min. Green: LC3B Polyclonal Antibody (Product # PA5-32254) diluted at 1:2,000. Red: alpha Tubulin, a cytoskeleton marker, stained by alpha Tubulin antibody. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunocytochemistry-Immunofluorescence analysis of LC3B was performed in HeLa cells fixed in 4% paraformaldehyde at RT for 15 min. Green: LC3B Polyclonal Antibody (Product # PA5-32254) diluted at 1:200. Red: phalloidin, a cytoskeleton marker. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemistry (Paraffin) analysis of LC3B was performed in paraffin-embedded mouse brain tissue using LC3B Polyclonal Antibody (Product # PA5-32254) at a dilution of 1:500.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

LC3B Polyclonal Antibody detects LC3B protein at cytoplasm by immunohistochemical analysis. Sample: Paraffin-embedded rat brain. LC3B stained by LC3B Polyclonal Antibody (Product # PA5-32254) diluted at 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

LC3B Polyclonal Antibody (Product # PA5-32254) detects LC3B protein by flow cytometry analysis. Sample: HeLa cell fixed in 4% paraformaldehyde at 4°C for 5 min. Brown: Unlabelled sample was also used as a control. Blue: LC3B Polyclonal Antibody (Product # PA5-32254) dilution: 1:100. Acquisition of >20,000 events were collected using Argon ion laser (488nm) and 525/30 bandpass filter.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunoprecipitation of LC3B was performed in U87-MG whole cell extracts using 5 µg of LC3B Polyclonal Antibody (Product # PA5-32254). Samples were transferred to a membrane and probed with LC3B Polyclonal Antibody as a primary antibody and an HRP-conjugated anti-Rabbit IgG was used as a secondary antibody.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 3 Low radiation environment switches pKZ1 A11 mouse hybridoma overgrowth cell response from apoptosis toward autophagy. (A) Western blots showing activation of p53 and induction of LC3B-II in LRE (LNGS)-grown pKZ1 A11 mouse hybridoma cells and activation of PARP1 and caspase 3 in RRE (ISS) grown pKZ1 A11 mouse hybridoma cells. (B) Densitometry analysis of western blots shown in (A) .