Class II HDAC Antibody Sampler Kit
cat.: HAK21073
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: P56524 Human | Q9UQL6 Human | Q9Z2V6 Mouse | Q9UBN7 Human | Q8WUI4 Human | Q8C2B3 Mouse | Q99P96 Rat | Q9UKV0 Human | Q969S8 Human | Q6P3E7 Mouse | Q569C4 Rat
Entrez Gene: 84580 Rat
Images
HAK21073_1.jpg Fig1: Western blot analysis of HDAC6 on different lysates with Rabbit anti-HDAC6 antibody (ET1701-66) at 1/1,000 dilution.

Lane 1: HCT 116-si NT cell lysate
Lane 2: HCT 116-si HDAC6 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 131 kDa
Observed band size: 160 kDa

Exposure time: 20 seconds; ECL: K1801;

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 (ET1701-66) 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.
HAK21073_2.jpg Fig2: Western blot analysis of HDAC10 on different lysates with Rabbit anti-HDAC10 antibody (ET1612-69) at 1/500 dilution.

Lane 1: Rat brain tissue lysate
Lane 2: Rat cerebellum tissue lysate
Lane 3: Rat pancreas tissue lysate

Lysates/proteins at 40 µg/Lane.

Predicted band size: 71 kDa
Observed band size: 75 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 (ET1612-69) at 1/500 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.
HAK21073_3.jpg Fig3: Western blot analysis of HDAC4 on different lysates with Rabbit anti-HDAC4 antibody (ET1612-51) at 1/1,000 dilution.

Lane 1: HCT 116-si NT cell lysate
Lane 2: HCT 116-si HDAC4 cell lysate

Lysates/proteins at 10 µg/Lane.
Predicted band size: 119 kDa
Observed band size: 150 kDa
Exposure time: 3 minutes; ECL: K1801;
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 (ET1612-51) 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.
HAK21073_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Rabbit anti-HDAC4 antibody (ET1612-51) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (ET1612-51) at 1/200 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.
HAK21073_5.jpg Fig5: HDAC5 was immunoprecipitated from 0.2 mg NIH/3T3 cell lysate with HA723022 at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using HA723022 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: NIH/3T3 cell lysate (input)
Lane 2: HA723022 IP in NIH/3T3 cell lysate
Lane 3: Rabbit IgG instead of HA723022 in NIH/3T3 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 5 seconds; ECL: K1801
HAK21073_6.jpg Fig6: Chromatin immunoprecipitations were performed with cross-linked chromatin from NIH/3T3 cells with HDAC5 (HA723022) or Normal Rabbit IgG according to the ChIP protocol. The enriched DNA was quantified by real-time PCR using indicated primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
HAK21073_7.jpg Fig7: Immunocytochemistry analysis of NIH/3T3 cells labeling HDAC5 with Rabbit anti-HDAC5 antibody (HA723022) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-HDAC5 antibody (HA723022) at 1/100 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 (HA601187, 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.
HAK21073_8.jpg Fig8: Immunohistochemical analysis of paraffin-embedded human testis tissue with Rabbit anti-HDAC4 antibody (ET1612-51) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (ET1612-51) at 1/200 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.
HAK21073_9.jpg Fig9: Immunocytochemistry analysis of HepG2 cells labeling HDAC5 with Rabbit anti-HDAC5 antibody (HA723022) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-HDAC5 antibody (HA723022) at 1/100 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 (HA601187, 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.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.