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XAB2 Polyclonal Antibody

Uniprot : Q9HCS7
  • Cat.No.:E-AB-53550

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

To Purchase E-AB-53550

Size:
  • 20μL
  • 60μL
  • 120μL
  • 200μL
Price: $69
Qty:

Test Application

  • Verified Samples

    Reactivity Application
    Human WB
    (293T,)

    Western Blot analysis of 293T cells using XAB2 Polyclonal Antibody at dilution of 1:250.

    IHC
    (colorectal cancer,liver cancer,)

    Immunohistochemistry of paraffin-embedded Human colorectal cancer tissue using XAB2 Polyclonal Antibody at dilution of 1:25(×200).

    Immunohistochemistry of paraffin-embedded Human liver cancer tissue using XAB2 Polyclonal Antibody at dilution of 1:25(×200).

  • Dilution

    WB 1:500-1:2000, IHC 1:25-1:100, ELISA 1:5000-1:10000

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 (Wet transfer)

1.Choose the PVDF Membrane (Cat# ) 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.Place the following materials in the order of the black plate (negative electrode) - fiber mat - filter paper - gel - PVDF Membrane - filter paper - fiber mat - white plate (positive electrode) are placed in order, discharge bubbles, clamp and place in the wet transfer tank. The recommended transmembrane conditions are . Make sure that the transmembrane process is carried out at low temperatures.
Note: This is for wet transfer. If other transmembrane methods are used, please adjust according to the specific conditions.

3.After the transmembrane, take out the PVDF Membrane carefully and wash with TBST Buffer for 1 min.

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 XAB2 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 Polyclonal
Isotype IgG
Concentration 0.4 mg/mL
Storage Store at -20℃. Avoid freeze / thaw cycles.
Buffer PBS with 0.05% NaN3 and 40% Glycerol,pH7.4
Purification Method Antigen affinity purification
Research Areas Epigenetics and Nuclear Signaling
Conjugation Unconjugated

Immunogen Details

Immunogen Synthetic peptide of human XAB2
Abbre XAB2
Synonyms 0610041O14Rik,adapter protein ATH 55,Adapter protein ATH-55,Ath55,AV025587,Crn related protein kim1 ,crn-related protein kim1,DKFZp762C1015,HCNP,HCNP protein,HCRN,KIAA1177,NTC90,PP3898,Pre-mRNA-splicing factor SYF1,Protein HCNP,RNA splicing factor,SYF1,SYF1 homolog,SYF1 homolog, RNA splicing factor ,SYF1 pre mRNA splicing factor,SYF1,Xab2,XPA binding protein 2,XPA-binding protein 2
Swissprot Q9HCS7
Gene Accession NP064581
Calculated MW 100 kDa
Observed MW Refer to figures

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. Detected in the splicing complex carrying pre-mRNA.

Background

HCNP, also known as XAB2 (Xeroderma pigmentosum group A (XPA) binding protein 2), HCRN, SYF1 or NTC90, is a nuclear protein that participates in transcription, transcription-coupled repair (TCR) and pre-mRNA splicing. It contains fifteen tetratricopeptide repeat motifs and associates with nucleotide excision repair machinery. More specifically, HCNP associates with Cockayne syndrome group A and B proteins (CSA and CSB), RNA Polymerase II (Pol II) and XPA in response to DNA damage and is believed to function in the TCR pathway. HCNP also functions as an essential component of a pre-mRNA splicing complex of the spliceosome (composed of AQR (aquarius), PRP19, CCDC16, HCNP, ISY1 and Cyclophilin E) and is required for proper RNA synthesis in the cell. In addition, HCNP functions as a component of the RAR corepressor complex with RARå and HDAC3 and exhibits an inhibitory effect on ATRA-induced cell differentiation. This suggests that HCNP may function as useful target in cancer therapy.

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