HAK21180

Wnt/Beta Catenin Signaling Pathway Antibody Sampler Kit

cat.: HAK21180

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.
Concentration:	1ug/ul
Uniprot #:	SwissProt: P01106 Human \| P01108 Mouse \| P09416 Rat \| P05412 Human \| P05627 Mouse \| P17325 Rat \| P24385 Human \| P25322 Mouse \| P39948 Rat \| Q9UJU2 Human \| P27782 Mouse \| Q9QXN1 Rat \| P08581 Human

Images

Fig1: Western blot analysis of Cyclin D1 on different lysates with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/5,000 dilution and competitor's antibody at 1/5,000 dilution.

Lane 1: MCF7 cell lysate
Lane 2: K-562 cell lysate (negative)
Lane 3: A431 cell lysate
Lane 4: Neuro-2a cell lysate
Lane 5: NIH/3T3 cell lysate
Lane 6: C6 cell lysate
Lane 7: SH-SY5Y cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 34 kDa
Observed band size: 35 kDa

Exposure time: 20 seconds; ECL: K1802;
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 (ET1601-31) at 1/5,000 dilution and competitor's antibody at 1/5,000 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.

Fig2: Western blot analysis of Cyclin D1 on different lysates with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/5,000 dilution.

Lane 1: MCF7-si NT cell lysate
Lane 2: MCF7-si Cyclin D1 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 34 kDa
Observed band size: 34 kDa

Exposure time: 17 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 (ET1601-31) at 1/5,000 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.

Fig3: Immunocytochemistry analysis of Neuro-2a cells labeling Cyclin D1 with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/2,000 dilution and competitor's antibody at 1/1,600 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-Cyclin D1 antibody (ET1601-31) at 1/2,000 dilution and competitor's antibody at 1/1,600 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.

	Fig4: Flow cytometric analysis of MCF7 cells labeling Cyclin D1. Cells were fixed and permeabilized. Then stained with the primary antibody (ET1601-31, red) at 1/5,000 dilution and competitor's antibody (red) at 1/2,000 dilution, compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
	Fig5: Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue using anti-Cyclin D1 antibody. 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 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1601-31, 1/200) for 30 minutes 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.
	Fig6: Application: IF-Tissue Species: Human Site: colon cancer Sample: Paraffin-embedded section Antibody concentration: 1/500

Fig7: Immunocytochemistry analysis of SH-SY5Y (positive) and 293T (negative) labeling Cyclin D1 with Rabbit anti-Cyclin D1 antibody (ET1601-31) at 1/2,000 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-Cyclin D1 antibody (ET1601-31) at 1/2,000 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.

Fig8: Western blot analysis of c-Myc on different lysates with Rabbit anti-c-Myc antibody (HA721182) at 1/1,000 dilution.

Lane 1: HEK-293 cell lysate
Lane 2: HeLa cell lysate
Lane 3: Jurkat cell lysate
Lane 4: K-562 cell lysate
Lane 5: Neuro-2a cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 49 kDa
Observed band size: 55 kDa

Exposure time: 1 minute; 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 (HA721182) 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.

	Fig9: c-Myc was immunoprecipitated from 0.2 mg HeLa cell lysate with HA721182 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA721182 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: HeLa cell lysate (input) Lane 2: HA721182 IP in HeLa cell lysate Lane 3: Rabbit IgG instead of HA721182 in HeLa cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 30 seconds; ECL: K1801
	Fig10: Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells with c-Myc (HA721182) 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.
	Fig11: Immunocytochemistry analysis of HEK-293 cells labeling c-Jun with Rabbit anti-c-Jun antibody (ET1608-3) at 1/100 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-c-Jun antibody (ET1608-3) 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 (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.

Fig12: c-Jun was immunoprecipitated from 0.2 mg NIH/3T3 cell lysate with ET1608-3 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1608-3 at 1/2,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: NIH/3T3 cell lysate (input)
Lane 2: ET1608-3 IP in NIH/3T3 cell lysate
Lane 3: Rabbit IgG instead of ET1608-3 in NIH/3T3 cell lysate

Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 46 seconds; ECL: K1801

Fig13: Immunocytochemistry analysis of Jurkat (positive) and HeLa (negative) labeling LEF1 with Rabbit anti-LEF1 antibody (HA721992) at 1/500 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-LEF1 antibody (HA721992) at 1/500 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.

Fig14: Western blot analysis of Met (C-Met) on different lysates with Rabbit anti-Met (C-Met) antibody (ET1606-45) at 1/1,000 dilution.

Lane 1: HCT 116-si NT cell lysate
Lane 2: HCT 116-si Met (C-Met) cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 156 kDa
Observed band size: 156 kDa

Exposure time: 2 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 (ET1606-45) 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.

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.