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CK-7 Polyclonal Antibody

  • Cat.No.:E-AB-40292

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

To Purchase E-AB-40292

Size:
  • 20μL
  • 60μL
  • 120μL
  • 200μL
Price: $65
Qty:

Test Application

  • Verified Samples

    Reactivity Application
    Human WB
    (A549,A431,Hela,)

    Western Blot analysis of A549 cells using CK-7 Polyclonal Antibody at dilution of 1:2000.

    Western Blot analysis of A431 cells and Hela cells using CK-7 Polyclonal Antibody at dilution of 1:2000.

    Western Blot analysis of A431 cells and Hela cells using CK-7 Polyclonal Antibody at dilution of 1:2000.

    IHC
    (liver,breast cancer,)

    Immunohistochemistry of paraffin-embedded Human liver using CK-7 Polyclonal Antibody at dilution of 1:100.

    Immunohistochemistry of paraffin-embedded Human breast cancer using CK-7 Polyclonal Antibody at dilution of 1:100.

    Rat IHC
    (liver,)

    Immunohistochemistry of paraffin-embedded Rat liver using CK-7 Polyclonal Antibody at dilution of 1:100.

    Mouse WB
    (lung,)

    Western Blot analysis of Mouse lung using CK-7 Polyclonal Antibody at dilution of 1:500.

  • Dilution

    WB 1:500-1:2000 IHC 1:100-1:200

  • Western Blot Operation Guide

    In order to facilitate the operation and ensure the accuracy of WB results, the Western Blot Detection kit (Cat# E-IR-R304) is now available, containing the reagents which are needed from sample preparation to result detection. Please order the appropriate kit according to your specific needs.

    Separating gel12%
    Blocking1.5 h
    Primary Antibody1:500-1:2000
    Secondary Antibody1:5000
    Click Here for More Details .. More ↓

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 12% 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 0.45 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 1.5 h.

2.According to the recommended primary antibody dilution ratio, use the TBST Buffer containing 5% Skim Milk Powder to dilute the CK-7 Antibody at 1:500-1:2000, 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 3 times, 15 min/time..

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 1:5000. Incubate at room temperature for 1 h on a shaker.

5.Wash the PVDF Membrane with TBST Buffer for 3 times, 15 min/time.

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 0.85 mg/mL
Storage Store at -20℃. Avoid freeze / thaw cycles.
Buffer PBS with 0.05% Proclin300 and 50% glycerol, pH7.4.
Purification Method Antigen Affinity Purification
Research Areas Cancer, Signal Transduction
Conjugation Unconjugated

Immunogen Details

Immunogen Recombinant Mouse Keratin, type II cytoskeletal 7 protein(PKSM041386)
Abbre CK-7
Synonyms CK 7, Cytokeratin 7, D15Wsu77e, K2C7, K7, Keratin 7, Keratin type II cytoskeletal 7, Krt2-7, MGC11625, Sarcolectin, Type II mesothelial keratin K7
Swissprot Q9DCV7
Gene ID 110310
Calculated MW 51 kDa
Observed MW 51 kDa
Cellular Localization Cytoplasm
Tissue Specificity Expressed in most simple epithelia tested including liver, lactating mammary gland, lung, kidney, stomach, duodenum, colon, oviduct, uterus, pancreas, epididymis, prostate, preputial gland and mesothelium, and in most stratified epithelia tested including dorsal skin, paw/toe, tail, tongue, cervix, forestomach, and bladder. Also expressed in Henle layer of the inner root sheath of whisker follicle.Highest expression level in urinary bladder urothelium.

Background

Keratins are a large family of proteins that form the intermediate filament cytoskeleton of epithelial cells,which are classified into two major sequence types. Type I keratins are a group of acidic intermediate filament proteins,including K9–K23,and the hair keratins Ha1–Ha8. Type II keratins are the basic or neutral courterparts to the acidic type I keratins,including K1–K8,and the hair keratins,Hb1–Hb6. KRT7,also named as cytokeratin 7,is one member of type II basic cytokeratin. It is specifically expressed in the simple epithelia lining the cavities of the internal organs and in the gland ducts and blood vessels,and their neoplasms. KRT7 is marker of epithelial tissues,but not present in carcinomas of stratified squamous cell origin.This antibody is specifically against KRT7.

Citations

  1. Cells (2021) IF: 6.6
    MUSE Stem Cells Can Be Isolated from Stromal Compartment of Mouse Bone Marrow, Adipose Tissue, and Ear Connective Tissue: A Comparative Study of Their In Vitro Properties

    DOI: 10.3390/cells10040761

    PMID: 33808472

    Sample: muscle tissue,liver,brain,MUSE cell

Reviews/Q&A

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  • Reviews
  • Q&A

Verified Customer

P*******fSubmitted [ Jan 10 2020 ]

  • Application:WB
  • Species:Human
  • Loading amount:10μg
  • Sample source:A431
  • Gel Running Conditions:Reduced, Denaturing, Other details:12%
  • Blocking:

    Blocking buffer:Milk

    Blocking concentration:5 %

    Blocking temperature:25℃

    Blocking time: 1hours 30minutes

  • Primary antibody:

    Dilution:1:1000

    Time: 17hours

  • Temperature:4℃

    Diluent:5% Milk

  • Secondary antibody:Use Elabscience secondary antibody
  • Dilution:1:5000
  • Detection:

    method:ECL

  • Description:Western Blot analysis of A431 cells using CK-7 Polyclonal Antibody at dilution of 1:1000.
Read More
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Verified Customer

N***********dSubmitted [ Jan 04 2020 ]

  • Application:WB
  • Species:Mouse
  • Loading amount:10μg
  • Sample source:Mouse lung
  • Gel Running Conditions:Reduced, Denaturing, Other details:12%
  • Blocking:

    Blocking buffer:Milk

    Blocking concentration:5 %

    Blocking temperature:25℃

    Blocking time: 1hours 30minutes

  • Primary antibody:

    Dilution:1:1000

    Time: 18hours

  • Temperature:4℃

    Diluent:5% Milk

  • Secondary antibody:Use Elabscience secondary antibody
  • Dilution:1:5000
  • Detection:

    method:ECL

  • Description:Western Blot analysis of Mouse lung using CK-7 Polyclonal Antibody at dilution of 1:1000.
Read More
Read Less

Verified Customer

Y***********iSubmitted [ Dec 10 2019 ]

  • Application:WB
  • Species:Human
  • Loading amount:10μg
  • Sample source:A549
  • Gel Running Conditions:Reduced, Denaturing, Other details:12%
  • Blocking:

    Blocking buffer:Milk

    Blocking concentration:5 %

    Blocking temperature:25℃

    Blocking time: 1hours 30minutes

  • Primary antibody:

    Dilution:1:1000

    Time: 17hours

  • Temperature:4℃

    Diluent:5% Milk

  • Secondary antibody:Use Elabscience secondary antibody
  • Dilution:1:5000
  • Detection:

    method:ECL

  • Description:Western Blot analysis of A549 cells using CK-7 Polyclonal Antibody at dilution of 1:1000.
Read More
Read Less
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