Autophagosome Marker Antibody Sampler Kit
cat.: HAK21022
Product Type: Antibody Sampler Kit
Species reactivity: Human
Applications: WB
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Uniprot #: SwissProt: O94817 Human | Q9H492 Human | Q91VR7 Mouse | Q6XVN8 Rat | Q9GZQ8 Human | Q9CQV6 Mouse | Q62625 Rat
Images
HAK21022_1.jpg Fig1: Western blot analysis of ATG12 on different lysates with Rabbit anti-ATG12 antibody (HA721504) at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HCT 116 cell lysate (20 µg/Lane)
Lane 3: HEK-293 cell lysate (20 µg/Lane)
Lane 4: SH-SY5Y cell lysate (20 µg/Lane)

Predicted band size: 15 kDa
Observed band size: 55/20 kDa

Exposure time: 3 minutes;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721504) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.
HAK21022_2.jpg Fig2: Western blot analysis of MAP1LC3A on different lysates with Rabbit anti-MAP1LC3A antibody (ET1611-37) at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HeLa treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 3: C2C12 cell lysate (20 µg/Lane)
Lane 4: C2C12 treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 5: C6 cell lysate (20 µg/Lane)
Lane 6: C6 treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)

Predicted band size: 14 kDa
Observed band size: 14/16 kDa

Exposure time: 24 seconds;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1611-37) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21022_3.jpg Fig3: Western blot analysis of LC3B on different lysates with Rabbit anti-LC3B antibody (ET1701-65) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HeLa treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 3: C2C12 cell lysate (20 µg/Lane)
Lane 4: C2C12 treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 5: C6 cell lysate (20 µg/Lane)
Lane 6: C6 treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 7: mouse brain tissue lysate (20 µg/Lane)
Lane 8: rat brain tissue lysate (20 µg/Lane)

Predicted band size: 14/16 kDa
Observed band size: 14/16 kDa

Exposure time: 3 minutes;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1701-65) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21022_4.png Fig4: Immunocytochemistry analysis of Hela cells treated with or without 50μM Chloroquine for 24 hours labeling LC3B with Rabbit anti-LC3B antibody (ET1701-65) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-LC3B antibody (ET1701-65) at 1/200 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HAK21022_5.jpg Fig5: Western blot analysis of LC3B on different lysates with Rabbit anti-LC3B antibody (ET1701-65) at 1/1,000 dilution.

Lane 1: HCT 116 cell lysate (20 µg/Lane)
Lane 2: HCT 116 treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 3: U-87 MG cell lysate (20 µg/Lane)
Lane 4: C6 cell lysate (20 µg/Lane)
Lane 5: Mouse brain tissue lysate (20 µg/Lane)
Lane 6: Rat brain tissue lysate (20 µg/Lane)

Predicted band size: 14/16 kDa
Observed band size: 14/16 kDa

Exposure time: 26 seconds;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1701-65) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21022_6.jpg Fig6: Fluorescence multiplex immunohistochemical analysis of human gastric cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-LC3B (ET1701-65, Green), anti-CD31 (M1511-8, Red) and anti-CA-IX (ET1701-51, Yellow) on human gastric cancer. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in three rounds of staining: in the order of ET1701-65 (1/100 dilution), M1511-8 (1/2,000 dilution) and ET1701-51 (1/100 dilution) for 20 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Olympus VS200 Slide Scanner.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.