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SPACA3 Polyclonal Antibody

Uniprot : Q8IXA5
  • Cat.No.:E-AB-52300

  • Host: Rabbit
  • Reactivity: H
  • Applications: IHC,ELISA

To Purchase E-AB-52300

Size:
  • 20μL
  • 60μL
  • 120μL
  • 200μL
Price: $73
Qty:

Test Application

  • Verified Samples

    Reactivity Application
    Human IHC
    (thyroid cancer,)

    Immunohistochemistry of paraffin-embedded Human thyroid cancer tissue using SPACA3 Polyclonal Antibody at dilution of 1:45(×200).

  • Dilution

    IHC 1:30-1:150, 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

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 SPACA3 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 1.5 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 Developmental biology, Immunology, Signal transduction
Conjugation Unconjugated

Immunogen Details

Immunogen Full length fusion protein
Abbre SPACA3
Synonyms 1700025M08Rik,ALLP17,Cancer/testis antigen 54,CT54,LYC3,Lysozyme like sperm specific secretory protein ALLP17,Lysozyme-like acrosomal sperm-specific secretory protein ALLP-17,Lysozyme-like protein 3,LYZL3,processed form,SACA3,SLLP1,SPACA3,Sperm acrosome associated 3,Sperm acrosome membrane-associated protein 3,Sperm lysozyme-like protein 1,Sperm protein reactive with antisperm antibodies,Sperm protein reactive with ASA
Swissprot Q8IXA5
Gene Accession BC029867
Cellular Localization Secreted and Cytoplasmic vesicle>secretory vesicle>acrosome membrane. Anterior acrosome in non-capacitated spermatozoa and retained in the equatorial segment and in the luminal face of both the inner and outer acrosomal membranes following capacitation and the acrosome reaction.

Background

SPACA3 (sperm acrosome associated 3), also known as sperm lysozyme-like protein 1, lysozyme-like protein 3, lysozyme-like acrosomal sperm-specific secretory protein ALLP-17, cancer/testis antigen 54 (CT54), LYC3, SPRASA or LYZL3, is a 215 amino acid protein that participates in the fusion and adhesion of sperm and egg plasma membrane during fertilization. Identified as a novel cancer/testis antigen in hematologic malignancies, SPACA3 has the ability to elicit B-cell immune responses in patients with cancer and is considered a potential target for immunotherapy. A member of the glycosyl hydrolase 22 family which is expressed in testis, placenta and epididymis, SPACA3 exists as two alternatively spliced isoforms; SPACA3 isoform 1 is a single-pass type II membrane protein of the sperm acrosome whereas SPACA3 isoform 2 is a secreted protein. Sperm surface membrane protein that may be involved in sperm-egg plasma membrane adhesion and fusion during fertilization. It could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane. The processed form has no detectable bacteriolytic activity in vitro.

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