Anti-ATG4B antibody [RP23040125]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ATG4B antibody [RP23040125] (AB308052)

For immunofluorescence analysis, 70% confluent log phase Rapamycin treated HeLa cells (treated with 100nM of Rapamycin A for 4 hours) were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature for detection of ATG14 using ab308052 at 2 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody at a dilution of 1/400 for 30 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with DAPI F-actin (Panel c : red) was stained with Alexa Fluor® 594 Phalloidin Panel d is a merged image showing nuclear localization Panel e shows untreated HeLa cells Panel f shows no primary antibody control The images were captured at 20X magnification

• WB

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Western blot - Anti-ATG4B antibody [RP23040125] (AB308052)

Western blot analysis of ATG4B was performed by loading 30 µg of HeLa (lane 1), HEK 293 (lane 2), Jurkat (lane 3), HCT 116 (lane 4), COS 7 (lane 5), MDCK (lane 6) and HepG2 (lane 7) cell lysates using a 4-12 % Bis-Tris gel electrophoresis system and pre-stained protein standard. Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature on a rocking platform. ATG4B was detected at ~44 kDa using ab308052 at 1-2 µg/mL in 5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody at 1 : 5000 dilution was used and chemiluminescent detection was performed (ECL).

All lanes:

Western blot - Anti-ATG4B antibody [RP23040125] (ab308052) at 1 µg/mL

Lane 1:

HeLa cell lysate at 30 µg

Lane 2:

HEK 293 cell lysate at 30 µg

Lane 3:

Jurkat cell lysate at 30 µg

Lane 4:

HCT-116 cell lysate at 30 µg

Lane 5:

Cos 7 cell lysate at 30 µg

Lane 6:

MDCK cell lysate at 30 µg

Lane 7:

HepG2 cell lysate at 30 µg

Secondary

All lanes:

HRP-conjugated Goat anti-Rabbit IgG Secondary Antibody at 1/5000 dilution

Observed band size: 44 kDa

true

• WB

Supplier Data

Western blot - Anti-ATG4B antibody [RP23040125] (AB308052)

Western blot was performed using ab308052 at 1 µg/mL and a 42 kDa band corresponding to ATG4B was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of Jurkat (Lane 1), PC-3 (Lane 2), MCF7 (Lane 3), NIH/3T3 (Lane 4), SK-BR-3 (Lane 5), MDA-MB-231 (Lane 6) and Daudi (Lane 7) were electrophoresed 4-12% Bis-Tris Protein Gel. Resolved proteins were then transferred onto a nitrocellulose membrane. The blot was probed with ab308052 and detected by chemiluminescence with a HRP-conjugated Goat anti-Rabbit IgG (H+L) Recombinant Secondary Antibody at 1 : 4000 dilution. Chemiluminescent detection was performed (ECL).

All lanes:

Western blot - Anti-ATG4B antibody [RP23040125] (ab308052) at 1 µg/mL

Lane 1:

Jurkat cells whole cell extract at 30 µg

Lane 2:

PC-3 whole cell extract at 30 µg

Lane 3:

MCF7 whole cell extract at 30 µg

Lane 4:

NIH/3T3 whole cell extract at 30 µg

Lane 5:

SK-BR-3 whole cell extract at 30 µg

Lane 6:

MDA-MB-231 whole cell extracts at 30 µg

Lane 7:

Daudi whole cell extract at 30 µg

Secondary

All lanes:

HRP-conjugated Goat anti-Rabbit IgG (H+L) Recombinant Secondary Antibody at 1/4000 dilution

Observed band size: 42 kDa

true