HSPA1A Polyclonal Antibody

Uniprot : P0DMV8
  • Cat.No.:E-AB-40302

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

To Purchase E-AB-40302

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

Test Application

  • Verified Samples

    Reactivity Application
    Human WB
    (HepG2,293T,K562,A431,A549,)

    Western Blot analysis of HepG2 cells, 293T cells, K562 cells, A431 cells and A549 cells using HSPA1A Polyclonal Antibody at dilution of 1:1000.

    Western Blot analysis of HepG2 cells, 293T cells, K562 cells, A431 cells and A549 cells using HSPA1A Polyclonal Antibody at dilution of 1:1000.

    Western Blot analysis of HepG2 cells, 293T cells, K562 cells, A431 cells and A549 cells using HSPA1A Polyclonal Antibody at dilution of 1:1000.

    Western Blot analysis of HepG2 cells, 293T cells, K562 cells, A431 cells and A549 cells using HSPA1A Polyclonal Antibody at dilution of 1:1000.

    Western Blot analysis of HepG2 cells, 293T cells, K562 cells, A431 cells and A549 cells using HSPA1A Polyclonal Antibody at dilution of 1:1000.

    IHC
    (liver,)

    Immunohistochemistry of paraffin-embedded Human liver using HSPA1A Polyclonal Antibody at dilution of 1:200.

    Rat WB
    (brain,)

    Western Blot analysis of Rat brain, Mouse liver and Mouse brain using HSPA1A Polyclonal Antibody at dilution of 1:1000.

    IHC
    (kidney,)

    Immunohistochemistry of paraffin-embedded Rat kidney using HSPA1A Polyclonal Antibody at dilution of 1:200.

    Mouse WB
    (liver,brain,)

    Western Blot analysis of Rat brain, Mouse liver and Mouse brain using HSPA1A Polyclonal Antibody at dilution of 1:1000.

    Western Blot analysis of Rat brain, Mouse liver and Mouse brain using HSPA1A Polyclonal Antibody at dilution of 1:1000.

  • Dilution

    WB 1:000-1:2000 IHC 1:100-1:300

  • Related Reagents (view more)

    Application Products
    WB Western Blot Detection Kit(E-IR-R304)
    IHC 2-step plus Poly-HRP Anti Mouse/Rabbit IgG Detection System (with DAB solution)(E-IR-R217)
  • Western Blot Operation Guide ( Download)

    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 gel10%
    Transfer Membrane150 mA, 2 h
    Blocking1.5 h
    Primary Antibody1:1000-1:2
    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 10% 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# 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.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 150 mA, 2 h. 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 1.5 h.

2.According to the recommended primary antibody dilution ratio, use the TBST Buffer containing 5% Skim Milk Powder to dilute the HSPA1A Antibody at 1:1000-1:2, 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

Isotype IgG
Concentration 1 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 Human Heat shock 70 kDa protein 1A protein(PKSH033820)
Abbre HSPA1A
Synonyms heat shock protein family A (Hsp70) member 1A,heat shock protein family A (Hsp70) member 1B,HSP70-1B,HSP70-2,HSP70.2
Swissprot P0DMV8
Gene ID 3303/3304
Calculated MW 70kDa
Observed MW 70kDa
Cellular Localization Nucleus.Cytoskeleton
Tissue Specificity Highest expression level in endothelial cell

Background

This intronless gene encodes a 70kDa heat shock protein which is a member of the heat shock protein 70 family. In conjuction with other heat shock proteins, this protein stabilizes existing proteins against aggregation and mediates the folding of newly translated proteins in the cytosol and in organelles. It is also involved in the ubiquitin-proteasome pathway through interaction with the AU-rich element RNA-binding protein 1. The gene is located in the major histocompatibility complex class III region, in a cluster with two closely related genes which encode similar proteins.

Reviews/Q&A

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  • Reviews
  • Q&A

Verified Customer

T*mSubmitted [ Nov 14 2019 ]

  • Application:WB
  • Species:Human
  • Loading amount:50μg
  • Sample source:A431
  • Gel Running Conditions:Reduced, Denaturing, Other details:10%
  • Blocking:

    Blocking buffer:Milk

    Blocking concentration:5 %

    Blocking temperature:25℃

    Blocking time: 1.5hours

  • Primary antibody:

    Dilution:1:1000

    Time: ~15hours

  • Temperature:4℃

    Diluent:5% skim milk

  • Secondary antibody:Use Elabscience secondary antibody E-AB-1003
  • Dilution:1:5000
  • Detection:

    method:ECL

  • Description:Strong antibody.It work good in A431 cell.The band is around 70kD.Could have diluted more.
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Verified Customer

T****rSubmitted [ Sep 23 2019 ]

  • Application:IHC
  • Species:Human
  • Sample source:Breast
  • Blocking:

    Blocking buffer:Serum

    Blocking concentration:5 %

    Blocking temperature:37℃

    Blocking time: 20minutes

  • Primary antibody:

    Dilution:1:200

    Time: 15hours

  • Temperature:4℃

    Diluent:PBS

  • Secondary antibody:Use Non-Elabscience secondary antibody DAKO(,HRP)
  • Description:To expose target proteins heat induced antigen retrieval was performed using 0.01M TE (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 5% Serum for 20 minutes at 37℃. Tissues were then probed at a dilution of 1/200 with Heat shock 70 kDa protein 1A overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBS and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin
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