Anti-O-Linked N-Acetylglucosamine antibody [EPR19847]

• IP

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Immunoprecipitation - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (AB202665)

O-Linked N-Acetylglucosamine was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with ab202665 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab202665 at 1/1,000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.
Lane 1 : HeLa whole cell lysate 10 μg (Input).
Lane 2 : ab202665 IP in HeLa whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab202665 in HeLa whole cell lysate.
Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (ab202665)

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• WB

Lab

Western blot - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (AB202665)

Blocking buffer and concentration 5% NFDM/TBST
Diluting buffer and concentration 5% NFDM/TBST

Ac45SGlcNAc, an inhibitor of OGT, decreases O-GlcNAc modification. Thiamet-G, an inhibitor of OGA, increases O-GlcNAc modification.

These two chemicals were kindly provided by our collaborator Dr. Xing Chen, Peking University.

The expression pattern is consistent with reference (PMID : 27716624)

All lanes:

Western blot - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (ab202665) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell) treated with DMSO (0.4%) as baseline control. whole cell lysate at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) treated with 200 μM Ac45SGlcNAc for 24 hours whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix adenocarcinoma epithelial cell) treated with 200 μM Thiamet-G for 24 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

Observed band size: 50-100 kDa

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Exposure time: 3s

• WB

Unknown

Western blot - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (AB202665)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure times : Lane 1 : 4 seconds; Lane 2 : 15 seconds.

All lanes:

Western blot - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (ab202665) at 1/1000 dilution

Lane 1:

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

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell) 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 1/100000 dilution

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• WB

Lab

Western blot - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (AB202665)

Blocking buffer and concentration : 5% NFDM/TBST
Diluting buffer and concentration : 5% NFDM/TBST

Exposure time :

Lane 1 : 8 seconds;
Lane 2 : 3 seconds;
Lane 3 : 26 seconds.

All lanes:

Western blot - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (ab202665) at 1/1000 dilution

Lane 1:

Rat brain lysate at 10 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

Lane 3:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 10 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

Observed band size: 20-100 kDa

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• Dot

Unknown

Dot Blot - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (AB202665)

Dot Blot - Anti-O-Linked N-Acetylglucosamine antibody [EPR19847] (ab202665 1 : 1000 dilution).

Lane 1 : O-linked N-acetylglucosamine (O-GlcNAc) peptide 10 ng.

Lane 2 : Non-O-GlcNAc peptide 10 ng.

Blocking/Dilution buffer : 5% NFDM/TBST.

Secondary : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051 1 : 100,000 dilution).

Exposure time of 3 minutes.

The peptides were kindly provided by our collaborator Dr. Xing Chen, Peking University.