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Recombinant YY1 Monoclonal Antibody

Uniprot : P25490
  • Cat.No.:E-AB-81623

  • Host: Rabbit
  • Reactivity: H,M,R
  • Applications: WB

To Purchase E-AB-81623

Size:
  • 50μL
  • 100μL
Price: $160
Qty:

Test Application

  • Verified Samples

    Reactivity Application
    Human WB
    (K562,Hela,)

    Western blot detection of YY1 in K562,Rat Brain,C6,3T3,Hela cell lysates using YY1 Rabbit mAb(1:1000 diluted).Predicted band size:45kDa.Observed band size:65kDa.

    Western blot detection of YY1 in K562,Rat Brain,C6,3T3,Hela cell lysates using YY1 Rabbit mAb(1:1000 diluted).Predicted band size:45kDa.Observed band size:65kDa.

    Rat WB
    (Rat Brain,C6,)

    Western blot detection of YY1 in K562,Rat Brain,C6,3T3,Hela cell lysates using YY1 Rabbit mAb(1:1000 diluted).Predicted band size:45kDa.Observed band size:65kDa.

    Western blot detection of YY1 in K562,Rat Brain,C6,3T3,Hela cell lysates using YY1 Rabbit mAb(1:1000 diluted).Predicted band size:45kDa.Observed band size:65kDa.

    Mouse WB
    (3T3,)

    Western blot detection of YY1 in K562,Rat Brain,C6,3T3,Hela cell lysates using YY1 Rabbit mAb(1:1000 diluted).Predicted band size:45kDa.Observed band size:65kDa.

  • Dilution

    WB 1:1000-1:2000

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 YY1 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

Clonality Rabbit Monoclonal
Isotype IgG
Concentration 300 μg/mL
Storage Store at -20℃. Avoid freeze / thaw cycles.
Buffer 50mM Tris-Glycine(pH 7.4), 0.15M NaCl, 40% Glycerol, 0.01% Sodium azide and 0.05% protective protein
Purification Method Affinity Purified
Research Areas Cancer, Epigenetics and Nuclear Signaling, Neuroscience
Clone No. R02-8A3
Conjugation Unconjugated

Immunogen Details

Immunogen A synthetic peptide of human YY1
Abbre YY1
Synonyms CF1,Delta,Delta transcription factor,INO80 complex subunit S,INO80S,NF E1,NF-E1,NFE1,OTTHUMP00000197459,Transcriptional repressor protein YY1,TYY1,UCR motif DNA binding protein,UCRBP,Yin and yang 1,Yin and Yang 1 protein,Yin Yang 1,Ying Yang 1,YY 1,YY 1 transcription factor,YY-1,YY1,YY1 transcription factor,Recombinant Rabbit mAb,Recombinant Rabbit Monoclonal Antibody
Swissprot P25490
Gene ID 7528
Calculated MW 45kDa
Observed MW 65kDa

Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size. The common factors include:

1. Post-translational modifications: For example, modifications such as glycosylation, phosphorylation, methylation, and acetylation will increase the molecular weight of the protein.

2. Splicing variants: Different expression patterns of various mRNA splicing bodies may produce proteins of different sizes.

3. Post-translational cleavage: Many proteins are first synthesized into precursor proteins and then cleaved to form active forms, such as COL1A1.

4. Relative charge: the composition of amino acids (the proportion of charged amino acids and uncharged amino acids).

5. Formation of multimers: For example, in protein dimer, strong interactions between proteins can cause the bands to be larger. However, the use of reducing conditions can usually avoid the formation of multimers.

If a protein in a sample has different modified forms at the same time, multiple bands may be detected on the membrane.

Cellular Localization Nucleus matrix. Associated with the nuclear matrix.

Background

YY1,also named as DELTA,INO80S and NF-E1,contains four C2H2-type zinc fingers and belongs to the YY transcription factor family. YY1 is a multifunctional transcription factor that exhibits positive and negative control on a large number of cellular and viral genes by binding to sites overlapping the transcription start site. YY1 may direct histone deacetylases and histone acetyltransferases to a promoter in order to activate or repress the promoter,thus implicating histone modification in the YY1. The open reading frame of the human YY1 cDNA encodes a protein of 414 amino acids with a predicted molecular weight of 44 kDa. However,YY1 migrates on SDS gels as a 65–68 kDa protein,probably due to the structure of the protein. It is a ubiquitously expressed transcription factor with fundamental roles in embryogenesis,differentiation,replication and proliferation.

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