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YAP1 Polyclonal Antibody

  • Cat.No.:E-AB-62344

  • Host: Rabbit
  • Reactivity: H,M,R
  • Applications: IHC,IF

To Purchase E-AB-62344

Size:
  • 60μL
  • 120μL
  • 200μL
Price: $200
Qty:

Test Application

  • Verified Samples

    Reactivity Application
    Human IHC
    (liver cancer,placenta,)

    Immunohistochemistry of paraffin-embedded Human liver cancer using YAP1 Polyclonal Antibody at dilution of 1:100 (40x lens).

    Immunohistochemistry of paraffin-embedded Human placenta using YAP1 Polyclonal Antibody at dilution of 1:100 (40x lens).

    Rat IHC
    (brain,)

    Immunohistochemistry of paraffin-embedded Rat brain using YAP1 Polyclonal Antibody at dilution of 1:100 (40x lens).

    IF
    (C6,)

    Immunofluorescence analysis of C6 cells using YAP1 Polyclonal Antibody at dilution of 1:100.Blue: DAPI for nuclear staining.

    Mouse IF
    (NIH/3T3,)

    Immunofluorescence analysis of NIH/3T3 cells using YAP1 Polyclonal Antibody at dilution of 1:100.Blue: DAPI for nuclear staining.

  • Dilution

    IHC 1:50-1:100 IF 1:50-1:200

Preparation of protein samples

1.Protein extraction

1)For tissue sample
a. Take the samples, wash the tissue thoroughly with pre-cooled PBS (0.01 M, pH=7.4)(Cat# E-BC-R187) to remove the surface blood and internal debris.
b. Weigh and smash the tissue, add an appropriate ratio of RIPA Lysis Buffer (Cat# E-BC-R327)(add 10 μL PMSF and 10 μL Na3VO4 to each 1 mL RIPA Lysis) and homogenizely lyse the tissue.
It is recommended to homogenize according to the ratio of tissue weight: RIPA volume = 3:10. For example, add 1 mL RIPA Lysis Buffer to 0.3 g tissue sample, the specific volume can be adjusted according to experimental requirements.
c. Shake and lyse on the ice for 30 min after homogenization. And then sonicate the sample for 1 min (under ice water bath conditions) with 2 s’ sonication and 2 s’ intervals to make cells fully lysis and reduce the viscosity of sample.
d. Centrifuge at 12,000 rpm for 10 min at 4℃.
e. Take the supernatant and measure the protein concentration mentioned in step2.

2)For cell sample
a. Collect the cells, wash them thoroughly with pre-cooled PBS (0.01 M, pH=7.4) to remove the medium off (it is generally recommended to wash 3 times).
b. Add an appropriate ratio of RIPA Lysate Buffer (10 μL PMSF and 10 μL Na3VO4 in each 1 mL RIPA Lysis) and lyse on the ice for 30 min.
It is recommended to add 0.1 mL of RIPA Lysis Buffer to each well of a 6-well plates (the protein content in different cells may vary, and the volume of the lysate added can be appropriately adjusted).
c. Sonicate the sample for 1 min (under ice water bath conditions) with 2 s’ sonication and 2 s’ intervals to make cells fully lyse and reduce viscosity of sample.
d. Centrifuge at 12,000 rpm for 10 min at 4℃.
e. Take the supernatant and measure the protein concentration mentioned in step2.

2.Measurement of protein concentration
By the BCA method (see the Total Protein Colorimetric Assay Kit (Cat# E-BC-K318) instructions).

3.Boiling the samples
Adjust the protein concentration with PBS Buffer. Add 5 × SDS Loading Buffer (Cat# E-BC-R288) with the ratio of the protein sample: 5 × SDS Loading Buffer = 4:1 and boil the mixture for 10 min. Centrifuge at 12,000 rpm for 2 min and collect the supernatant. The denatured protein can be employed to Western Blot experiments or stored at -20℃ or -80℃.

Note: It is recommended that the total protein loading amount of test sample is about 50 μg in each well. Try to make the loading volume of each sample close to 10 μL.

Electrophoresis

1.According to the molecular weight of the target protein, prepare 0% separation gel. Add the test sample to each well, and add 5 μL of Pre-stained Protein Marker (Cat# E-BC-R273)to a reserved well in order to verify the target molecular weight and the extent of membrane transfer. Add Electrophoresis Buffer ( Cat# E-BC-R331) and start electrophoresis.

2.Electrophoresis at 80v when the samples are in stacking gel, then convert to 120v when the blue flow into the separating gel. Electrophoresis time is about 2-3 h till bromophenol blue reaches the bottom of the gel.

Transfer Membrane

1.Choose the PVDF Membrane (Cat# E-BC-R266) with a pore size of μm according to the molecular weight of the target protein. Soak the PVDF Membrane in methanol for 1 min to activate it, and then soak the PVDF Membrane in the Transmembrane Buffer (Cat# E-BC-R333), the filter paper and fiber mat must be soaked in the Transmembrane Buffer for use too.

2.Follow manufacture instructions of Transfer System for wet, semi-dry, or dry transfer.

Incubation of antibodies

1.Soak the PVDF Membrane with TBST Buffer (Cat# E-BC-R335) containing 5% Skim Milk Powder as blocking buffer and block the membrane at room temperature for .

2.According to the recommended primary antibody dilution ratio, use the TBST Buffer containing 5% Skim Milk Powder to dilute the YAP1 Antibody at , soak the PVDF Membrane in the primary antibody working solution, incubate overnight at 4 ℃, and gently shake.

3.Wash the PVDF Membrane with TBST Buffer for .

4.According to the recommended secondary antibody dilution ratio, use a TBST Buffer solution containing 2% Skim Milk Powder to dilute Goat Anti-Rabbit IgG (H+L) (peroxidase/HRP conjugated) (Cat# E-AB-1003) at . Incubate at room temperature for 1 h on a shaker.

5.Wash the PVDF Membrane with TBST Buffer for .

Detection

1.Mix A and B in the Excellent Chemiluminescent Substrate Detection kit (Cat# E-BC-R347) at the ratio of 1:1 as working solution.

2.Take out the PVDF Membrane from TBST Buffer and absorb the liquid with the filter paper. Pave the PVDF Membrane on the detection machine, add ECL working solution continuously on the PVDF Membrane, discharge the bubble and detect the result.

3.Adjust the contrast and the exposure time to get the best image.

Appendix

Product Details

Isotype IgG
Concentration 1mg/mL
Storage Store at -20°C. Avoid freeze / thaw cycles.
Buffer PBS with 0.02% sodium azide, 50% glycerol, pH7.3.
Purification Method Affinity purification
Research Areas Cancer, Epigenetics and Nuclear Signaling, Signal Transduction
Conjugation Unconjugated

Immunogen Details

Immunogen Recombinant fusion protein of human YAP1 (NP_001123617.1).
Abbre YAP1
Synonyms COB1,YAP,YAP2,YAP65,YKI,YAP1
Swissprot P46937
Gene ID 10413
Cellular Localization Cytoplasm. Nucleus. Both phosphorylation and cell density can regulate its subcellular localization. Phosphorylation sequesters it in the cytoplasm by inhibiting its translocation into the nucleus. At low density, predominantly nuclear and is translocated to the cytoplasm at high density.
Tissue Specificity Increased expression seen in some liver and prostate cancers. Isoforms lacking the transactivation domain found in striatal neurons of patients with Huntington disease (at protein level).

Background

This gene encodes a downstream nuclear effector of the Hippo signaling pathway which is involved in development, growth, repair, and homeostasis. This gene is known to play a role in the development and progression of multiple cancers as a transcriptional regulator of this signaling pathway and may function as a potential target for cancer treatment. Alternative splicing results in multiple transcript variants encoding different isoforms.

Citations

  1. NATURE IMMUNOLOGY (2022) IF: 25.606
    Neutrophils direct preexisting matrix to initiate repair in damaged tissues

    DOI: 10.1038/s41590-022-01166-6

    PMID: 35354953

    Sample: peritoneal matrix

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