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CD146 Ployclonal Antibody

Uniprot : Q9EPF2
  • Cat.No.:E-AB-40373

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

To Purchase E-AB-40373

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

Test Application

  • Verified Samples

    Reactivity Application
    Rat IHC
    (lung,)

    Immunohistochemistry of paraffin-embedded Rat lung using CD146 Ployclonal Antibody at dilution of 1:200.

    Mouse IHC
    (heart,lung,)

    Immunohistochemistry of paraffin-embedded Mouse heart using CD146 Ployclonal Antibody at dilution of 1:200.

    Immunohistochemistry of paraffin-embedded Mouse lung using CD146 Ployclonal Antibody at dilution of 1:200.

  • Dilution

    IHC 1:100-1:300

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 CD146 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 0.61mg/mL
Storage Store at -20℃. Avoid freeze / thaw cycles.
Buffer PBS with 0.05% Proclin300, 50% glycerol, pH7.3.
Purification Method Antigen Affinity Purification
Research Areas Immunology, Signal Transduction, Cancer, Stem Cells, Cardiovascular
Conjugation Unconjugated

Immunogen Details

Immunogen Recombinant Rat Cell surface glycoprotein MUC18 protein
Abbre CD146
Synonyms A32 antigen,CD 146,CD146,CD146 antigen,Cell surface glycoprotein MUC18,Cell surface glycoprotein P1H12,Gicerin,Mcam,Melanoma adhesion molecule,Melanoma associated antigen A32,Melanoma associated antigen MUC18,Melanoma associated glycoprotein MUC18,Melanoma cell adhesion molecule,Melanoma-associated antigen A32,Melanoma-associated antigen MUC18,MelCAM,MUC 18,MUC18,MUC18,S endo 1,S endo 1 endothelial associated antigen,S-endo 1 endothelial-associated antigen
Swissprot Q9EPF2
Gene ID 78967
Cellular Localization Cell membrane, Membrane
Tissue Specificity Detected in lung, uterus and placenta (at protein level). Detected in heart, lung, kidney, adrenal gland, intestine, testis, skeletal muscle and aorta. Detected at low levels in adult brain, in particular in brain stem and spinal cord, but also in hippocampus, olfactory bulb and striatum (at protein level).

Background

CD146,also known as melanoma cell adhesion molecule (MCAM) or MUC18,originally identified as a biomarker of melanoma progression,is a transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily . Structurally,it consists of five Ig domains,a transmembrane domain,and a cytoplasmic region. In normal adult tissue,CD146 is primarily expressed by vascular endothelium and smooth muscle. CD146 is a key cell adhesion protein in vascular endothelial cell activity and angiogenesis,and has been used as marker of circulating endothelium cells (CECs) .

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