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    hsp70-antibody-3a3-ab5439.pdf

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Signal Transduction Protein Trafficking Chaperones Heat Shock Proteins
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Anti-Hsp70 antibody [3A3] (ab5439)

  • Datasheet
  • SDS
Reviews (3)Q&A (6)References (33)

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Western blot - Anti-Hsp70 antibody [3A3] (ab5439)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)
  • Western blot - Anti-Hsp70 antibody [3A3] (ab5439)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [3A3] (ab5439)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [3A3] (ab5439)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [3A3] (ab5439)
  • Immunoprecipitation - Anti-Hsp70 antibody [3A3] (ab5439)
  • Flow Cytometry - Anti-Hsp70 antibody [3A3] (ab5439)

Key features and details

  • Mouse monoclonal [3A3] to Hsp70
  • Suitable for: Flow Cyt, ICC/IF, IHC-P, WB, IP
  • Reacts with: Mouse, Rat, Cow, Human, African green monkey
  • Isotype: IgG1

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Overview

  • Product name

    Anti-Hsp70 antibody [3A3]
    See all Hsp70 primary antibodies
  • Description

    Mouse monoclonal [3A3] to Hsp70
  • Host species

    Mouse
  • Specificity

    ab5439 detects several members of the heat shock protein 70 kDa (Hsp 70) gene family including Hsp 70, Hsc 70, p75, and following heat shock, Hsp 72 from yeast, Drosophila, fish, mouse, avian, amphibian and human samples.
  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Mouse
    Human
    IHC-P
    Human
    IP
    Human
    WB
    Human
    See all applications and species data
  • Immunogen

    Recombinant fragment corresponding to Human Hsp70 aa 504-617 (C terminal).

  • Epitope

    Epitope mapping with a panel of Hsp 70 deletion mutants suggests that the epitope recognized is located between amino acids 504-617 of human Hsp 70, a region that has been shown to be involved in stress-induced nucleolar localization.
  • Positive control

    • ICC/IF: Heat shocked MCF7, HeLa and NIH-3T3, HEK-293 cells. WB: HEK-293, A549, IMR32, Jurkat, Jurkat whole cell lysate treated with heat shock, MDBK, NIH/3T3, PC-12, COS-7, HeLa, F9 cell lysates. Mouse ovary tissue lysates. IHC-P: Human breast carcinoma, testis and tonsil tissues. Flow Cyt: Jurkat cells. IP: HeLa whole cell lysates.
  • General notes

     This product is in progress to switch to a recombinant production method. Recombinant stock will be available end of March 2021. 

     

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    Preservative: 0.05% Sodium azide
    Constituent: 99% PBS
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Primary antibody notes

    The Hsp 70 family is a set of highly conserved proteins that are induced by a variety of biological stresses, including heat stress, in every organism in which the proteins have been examined. The human Hsp 70 family members include: Hsp 70, a protein which is strongly inducible in all organisms but which is also constitutively expressed in primate cells; Hsp 72, a 72 kDa protein that is induced exclusively under stress conditions; Hsc 70, or cognate protein, is a 72 kDa, constitutively expressed, protein which is involved in the uncoating of clathrin coated vesicles; GRP78, or BiP, is a glucose regulated 78 kDa protein localized in the endoplasmic reticulum; and p75, or Hsp 75, a 75 kDa protein that is found within the mitochondria.
  • Clonality

    Monoclonal
  • Clone number

    3A3
  • Isotype

    IgG1
  • Research areas

    • Signal Transduction
    • Protein Trafficking
    • Chaperones
    • Heat Shock Proteins
    • Cancer
    • Tumor biomarkers
    • Other

Associated products

  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgG H&L (HRP) (ab205719)
    • Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879)
  • Isotype control

    • Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control (ab170190)
    • Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control (ab91353)
  • Recombinant Protein

    • Recombinant human Hsp70 protein (Active) (ab78434)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab5439 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
Flow Cyt
Human
ICC/IF
Mouse
Human
IHC-P
Human
IP
Human
WB
Human
Application Abreviews Notes
Flow Cyt
1/100.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC/IF
1/50 - 1/200.
IHC-P
1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
WB (3)
1/1000 - 1/5000. Predicted molecular weight: 70-78 kDa.

Representing different members of the HSP70 family. 2-dimensional gel electrophoresis is required to resolve the heat induced form of these proteins from their constitutively expressed counterparts.

IP
Use a concentration of 2 µg/ml.
Notes
Flow Cyt
1/100.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC/IF
1/50 - 1/200.
IHC-P
1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
WB
1/1000 - 1/5000. Predicted molecular weight: 70-78 kDa.

Representing different members of the HSP70 family. 2-dimensional gel electrophoresis is required to resolve the heat induced form of these proteins from their constitutively expressed counterparts.

IP
Use a concentration of 2 µg/ml.

Target

  • Relevance

    Function: In cooperation with other chaperones, the Hsp70 family stabilize preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. These chaperones participate in all these processes through their ability to recognize nonnative conformations of other proteins. They bind extended peptide segments with a net hydrophobic character exposed by polypeptides during translation and membrane translocation, or following stress-induced damage. In case of rotavirus A infection, serves as a post-attachment receptor for the virus to facilitate entry into the cell. Tissue specificity: HSPA1B is testis-specific.
  • Cellular localization

    Cytoplasm. Localized in cytoplasmic mRNP granules containing untranslated mRNAs.
  • Database links

    • Entrez Gene: 281825 Cow
    • Entrez Gene: 15511 Human
    • Entrez Gene: 3303 Human
    • Entrez Gene: 193740 Mouse
    • Entrez Gene: 3304 Mouse
    • Entrez Gene: 24472 Rat
    • Entrez Gene: 294254 Rat
    • Omim: 140550 Human
    • Omim: 603012 Human
    • SwissProt: Q27975 Cow
    • SwissProt: P0DMV8 Human
    • SwissProt: P0DMV9 Human
    • SwissProt: P17879 Mouse
    • SwissProt: Q61696 Mouse
    • SwissProt: P0DMW0 Rat
    • SwissProt: P0DMW1 Rat
    see all
  • Alternative names

    • DnaK type molecular chaperone HSP70 1 antibody
    • Epididymis secretory protein Li 103 antibody
    • FLJ54303 antibody
    • FLJ54370 antibody
    • FLJ54392 antibody
    • FLJ54408 antibody
    • FLJ75127 antibody
    • Heat shock 70 kDa protein 1 antibody
    • Heat shock 70 kDa protein 1/2 antibody
    • Heat shock 70 kDa protein 1A antibody
    • Heat shock 70 kDa protein 1A/1B antibody
    • Heat shock 70kDa protein 1B antibody
    • Heat shock induced protein antibody
    • HEL S 103 antibody
    • HSP70 1 antibody
    • HSP70 1A antibody
    • HSP70 1B antibody
    • HSP70 2 antibody
    • HSP70-1 antibody
    • HSP70-1/HSP70-2 antibody
    • HSP70-1A antibody
    • HSP70-2 antibody
    • HSP70.1 antibody
    • HSP70.1/HSP70.2 antibody
    • HSP71_HUMAN antibody
    • HSP72 antibody
    • HSPA1 antibody
    • HSPA1A antibody
    • HSPA1B antibody
    see all

Images

  • Western blot - Anti-Hsp70 antibody [3A3] (ab5439)
    Western blot - Anti-Hsp70 antibody [3A3] (ab5439)
    All lanes : Anti-Hsp70 antibody [3A3] (ab5439) at 1/1000 dilution

    Lane 1 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate
    Lane 2 : A549 (human lung carcinoma cell line) whole cell lysate
    Lane 3 : IMR32 (human brain neuroblast cell line) whole cell lysate
    Lane 4 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate
    Lane 5 : Jurkat whole cell lysate treated with heat shock (42deg for 30 min followed by 37deg for 3 hours)
    Lane 6 : MDBK (bovine kidney cell line) whole cell lysate
    Lane 7 : NIH/3T3 (mouse embryonic fibroblast cell line) whole cell lysate
    Lane 8 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate
    Lane 9 : COS-7 (african green monkey kidney fibroblast-like cell line) whole cell lysate
    Lane 10 : Mouse ovary tissue lysate

    Secondary
    All lanes : Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP at 1/4000 dilution

    Predicted band size: 70-78 kDa

  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)

    Immunofluorescence analysis of HEK-293 (human epithelial cell line from embryonic kidney) cells labeling HSP70 with ab5439 at 1/200 dilution in 0.1% BSA, incubated at 4°C overnight, followed by Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 conjugate at 1/2000 dilution for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (Panel c: red) was stained with Rhodamine Phalloidin at 1/300 dilution. Panel d represents the merged image showing localization to nucleus and cytoplasm. Panel e represents control cells with no primary antibody to assess background.

    The cells (70% confluent log phase) were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The images were captured at 60X magnification.

  • Western blot - Anti-Hsp70 antibody [3A3] (ab5439)
    Western blot - Anti-Hsp70 antibody [3A3] (ab5439)
    All lanes : Anti-Hsp70 antibody [3A3] (ab5439) at 1 µg/ml

    Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate
    Lane 3 : NIH/3T3 (mouse embryonic fibroblast cell line) whole cell lysate
    Lane 4 : F9 (mouse embryonic testicular cancer cell line) whole cell lysate
    Lane 5 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 70-78 kDa
    Observed band size: 73 kDa
    why is the actual band size different from the predicted?

  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)

    Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) ab5439 shows staining in MCF7 (human breast adenocarcinoma cell line) cells. Heat Shock Protein 70 staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Heat Shock Protein 70 ab5439 at a dilution of 1/100-1/200 over night at 4 oC washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)

    Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) ab5439shows staining in HeLa (human epithelial cell line from cervix adenocarcinoma) cells. Heat Shock Protein 70 staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Heat Shock Protein 70 ab5439 at a dilution of 1/100-1/200 over night at 4oC washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp70 antibody [3A3] (ab5439)

    Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) ab5439 shows staining in NIH/3T3 (mouse embryonic fibroblast cell line) cells. Heat Shock Protein 70 staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Heat Shock Protein 70 ab5439 at a dilution of 1/100-1/200 over night at 4oC washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [3A3] (ab5439)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [3A3] (ab5439)

    Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human breast carcinoma tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/50 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab5439) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [3A3] (ab5439)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [3A3] (ab5439)

    Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human testis tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/200 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab5439) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [3A3] (ab5439)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsp70 antibody [3A3] (ab5439)

    Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/20 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab5439) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Immunoprecipitation - Anti-Hsp70 antibody [3A3] (ab5439)
    Immunoprecipitation - Anti-Hsp70 antibody [3A3] (ab5439)

    Immunoprecipitation of Hsp70 was performed on HeLa (human epithelial cell line from cervix adenocarcinoma) cells. Antigen:antibody complexes were formed by incubating 500ug whole cell lysate with 2ul of Hsp70 monoclonal antibody (ab5439) overnight on a rocking platform at 4oC. The immune complexes were captured on 50ul Protein Agarose washed extensively and eluted with Buffer. Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel and transferred to a PVDF membraneand blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Hsp70 monoclonal antibody (ab5439) at a dilution of 1/1000 overnight rotating at 4oC and probed with goat anti-mouse IgG-HRP secondary antibody at a dilution of 1/20,000 for at least 1 hour. Chemiluminescent detection was performed.

  • Flow Cytometry - Anti-Hsp70 antibody [3A3] (ab5439)
    Flow Cytometry - Anti-Hsp70 antibody [3A3] (ab5439)

    Overlay histogram showing Jurkat (human T cell leukemia cell line from peripheral blood) cells stained with ab5439 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab5439, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Protocols

  • Flow cytometry protocols
  • Immunoprecipitation protocols
  • Immunohistochemistry protocols
  • Immunocytochemistry & immunofluorescence protocols
  • Western blot protocols

Click here to view the general protocols

Datasheets and documents

    • Datasheet
    • SDS
  • References (33)

    Publishing research using ab5439? Please let us know so that we can cite the reference in this datasheet.

    ab5439 has been referenced in 33 publications.

    • Kong F  et al. Cold Exposure-Induced Up-Regulation of Hsp70 Positively Regulates PEDV mRNA Synthesis and Protein Expression In Vitro. Pathogens 9:N/A (2020). PubMed: 32224931
    • Adriaenssens E  et al. BAG3 Pro209 mutants associated with myopathy and neuropathy relocate chaperones of the CASA-complex to aggresomes. Sci Rep 10:8755 (2020). PubMed: 32472079
    • Hu W  et al. Effects of transport stress on pathological injury and expression of main heat shock proteins in the caprine stomach. BMC Vet Res 16:347 (2020). PubMed: 32962700
    • Fukuta T  et al. Low level electricity increases the secretion of extracellular vesicles from cultured cells. Biochem Biophys Rep 21:100713 (2020). PubMed: 31828227
    • Hancock ML  et al. Insulin Receptor Associates with Promoters Genome-wide and Regulates Gene Expression. Cell 177:722-736.e22 (2019). PubMed: 30955890
    View all Publications for this product

    Customer reviews and Q&As

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    1-3 of 3 Abreviews or Q&A

    Western blot abreview for Anti-Hsp70 antibody [3A3]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (cell lysates in 25% Nupage LSD, 10% DTT, 65% milli)
    Gel Running Conditions
    Reduced Denaturing (12.5% Acrylamide gel)
    Loading amount
    35 µg
    Specification
    cell lysates in 25% Nupage LSD, 10% DTT, 65% milli
    Blocking step
    Rockland blocking buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 20°C
    Read More

    Abcam user community

    Verified customer

    Submitted May 27 2015

    Western blot abreview for Anti-Hsp70 [3A3] antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    20 µg
    Gel Running Conditions
    Reduced Denaturing (8% acrylamide)
    Sample
    Pig Tissue lysate - other (Muscle sarcoplasmic extract)
    Specification
    Muscle sarcoplasmic extract
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More

    Dr. Shannon Cruzen

    Verified customer

    Submitted Mar 27 2014

    Western blot abreview for Anti-Hsp70 [3A3] antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Loading amount
    30 µg
    Gel Running Conditions
    Reduced Denaturing (12% Tris Glycine)
    Sample
    Human Cell lysate - whole cell (U2OS and HeLa cells)
    Specification
    U2OS and HeLa cells
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Jan 18 2011

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