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☑ Cell treatment (CT)
Western blot analysis of Pan-Lactyl-lysine on different lysates with Rabbit anti-Pan-Lactyl-lysine antibody (HA750908) at 1/2,000 dilution.
Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 100mM Lactate sodium for 24 hours cell lysate
Lysates/proteins at 20 µg/Lane.
Observed band size: Mutiple kDa
Exposure time: 59 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 (HA750908) at 1/2,000 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.
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☑ Cell treatment (CT)
Immunocytochemistry analysis of HeLa cells treated with or without 100mM Lactate sodium for 24 hours labeling Pan-Lactyl-lysine with Rabbit anti-Pan-Lactyl-lysine antibody (HA750908) at 1/1,000 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Pan-Lactyl-lysine antibody (HA750908) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. 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.
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Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-Pan-Lactyl-lysine antibody (HA750908) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750908) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Rabbit anti-Pan-Lactyl-lysine antibody (HA750908) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750908) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Immunohistochemical analysis of paraffin-embedded mouse small intestine tissue with Rabbit anti-Pan-Lactyl-lysine antibody (HA750908) at 1/500 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750908) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Immunohistochemical analysis of paraffin-embedded rat small intestine tissue with Rabbit anti-Pan-Lactyl-lysine antibody (HA750908) at 1/500 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750908) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Dot blot analysis of Pan-Lactyl-lysine on different proteins with Rabbit anti-Pan-Lactyl-lysine antibody (HA750908) at 1/2,000 dilution. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution for 1 hour at room temperature.
Lane 1: Lac pepetide library (positive)
Lane 2: Ac peptide library (negative)
Lane 3: Non-lac peptide library (negative)
Proteins loading: 100ng, 20ng, 4ng;
Blocking and dilution buffer: 5% NFDM/TBST;
Exposure time: 20 seconds.
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