Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Hsc70 antibody [EP1531Y] (ab51052)

Reviews (2)Q&A (3)References (22)
Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsc70 antibody [EP1531Y] (ab51052)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsc70 antibody [EP1531Y] (ab51052)
  • Flow Cytometry - Anti-Hsc70 antibody [EP1531Y] (ab51052)
  • Immunoprecipitation - Anti-Hsc70 antibody [EP1531Y] (ab51052)
  • Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
  • Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
  • Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
  • Anti-Hsc70 antibody [EP1531Y] (ab51052)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP1531Y] to Hsc70
  • Suitable for: Flow Cyt, IP, WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-Hsc70 antibody [EP1531Y]
    See all Hsc70 primary antibodies

  • Description

    Rabbit monoclonal [EP1531Y] to Hsc70

  • Host species

    Rabbit

  • Tested Applications & Species

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

  • Immunogen

    Synthetic peptide within Human Hsc70 aa 600 to the C-terminus (C terminal). The exact sequence is proprietary.

  • Positive control

    • WB: A431, MCF7, and HeLa cell lysates. IHC-P: Human cerebral cortex tissue. ICC/IF: MCF7 cells. Flow Cyt: MCF7 cells. IP: HeLa cell lysate.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

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

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    EP1531Y

  • Isotype

    IgG

  • Research areas

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab51052 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
Human
IHC-P
Human
IP
Human
WB
Mouse
Rat
Human
Application Abreviews Notes
Flow Cyt
1/30 - 1/70.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP (1)
1/10 - 1/20.
WB
1/500 - 1/5000. Detects a band of approximately 71 kDa (predicted molecular weight: 71 kDa).
IHC-P
1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.
ICC/IF (1)
1/100 - 1/250.
Notes
Flow Cyt
1/30 - 1/70.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP
1/10 - 1/20.
WB
1/500 - 1/5000. Detects a band of approximately 71 kDa (predicted molecular weight: 71 kDa).
IHC-P
1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.
ICC/IF
1/100 - 1/250.

Target

  • Function

    Acts as a repressor of transcriptional activation. Inhibits the transcriptional coactivator activity of CITED1 on Smad-mediated transcription. Chaperone. Isoform 2 may function as an endogenous inhibitory regulator of HSC70 by competing the co-chaperones.

  • Tissue specificity

    Ubiquitous.

  • Sequence similarities

    Belongs to the heat shock protein 70 family.

  • Domain

    The N-terminal 1-386 residues constitute the ATPase domain, while residues 387-646 form the peptide-binding domain.

  • Post-translational
    modifications

    Phosphorylated upon DNA damage, probably by ATM or ATR.
    ISGylated.

  • Cellular localization

    Cytoplasm. Melanosome. Localized in cytoplasmic mRNP granules containing untranslated mRNAs. Translocates rapidly from the cytoplasm to the nuclei, and especially to the nucleoli, upon heat shock. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • 2410008N15Rik antibody
    • Constitutive heat shock protein 70 antibody
    • Epididymis luminal protein 33 antibody
    • Epididymis secretory sperm binding protein Li 72p antibody
    • Heat shock 70 kDa protein 8 antibody
    • Heat shock 70kD protein 10 antibody
    • Heat shock 70kD protein 8 antibody
    • Heat shock 70kDa protein 8 antibody
    • Heat shock cognate 71 kDa protein antibody
    • Heat shock cognate protein 54 antibody
    • Heat shock cognate protein 71 kDa antibody
    • Heat shock protein 8 antibody
    • Heat shock protein A8 antibody
    • Heat shock protein family A (Hsp70) member 8 antibody
    • Heat-shock70-KD protein 10, formerly antibody
    • HEL 33 antibody
    • HEL S 72p antibody
    • HSC54 antibody
    • HSC71 antibody
    • Hsc73 antibody
    • HSP71 antibody
    • HSP73 antibody
    • HSP7C_HUMAN antibody
    • HSPA10 antibody
    • HSPA8 antibody
    • LAP1 antibody
    • Lipopolysaccharide associated protein 1 antibody
    • LPS associated protein 1 antibody
    • LPS associated protein antibody
    • MGC102007 antibody
    • MGC106514 antibody
    • MGC114311 antibody
    • MGC118485 antibody
    • MGC131511 antibody
    • MGC29929 antibody
    • N-myristoyltransferase inhibitor protein 71 antibody
    • NIP71 antibody
    see all

Images

  • Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    All lanes : Anti-Hsc70 antibody [EP1531Y] (ab51052) at 1/500 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : HSPA8 knockout HeLa cell lysate
    Lane 3 : A431 cell lysate
    Lane 4 : MCF7 cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 71 kDa
    Observed band size: 71 kDa



    Lanes 1- 4: Merged signal (red and green). Green - ab51052 observed at 71 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab51052 was shown to react with Hsc70 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265664 (knockout cell lysate ab256944) was used. Wild-type HeLa and HSPA8 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab51052 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 500 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Immunocytochemistry/ Immunofluorescence - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsc70 antibody [EP1531Y] (ab51052)

    Immunofluorescence staining of MCF7 cells with purified ab51052 at a working dilution of 1/250, counter-stained with DAPI. The secondary antibody was Alexa Fluor® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab51052 was used at a dilution of 1/500 followed by an Alexa Fluor® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Immunohistochemical staining of paraffin embedded human cerebral cortex with purified ab51052 at a working dilution of 1/500. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
  • Flow Cytometry - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Flow Cytometry - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Overlay histogram showing MCF7 cells fixed in 4% PFA and stained with purified ab51052 at a dilution of 1 in 70 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).
  • Immunoprecipitation - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Immunoprecipitation - Anti-Hsc70 antibody [EP1531Y] (ab51052)

    ab51052 (purified) at 1/20 immunoprecipitating Hsc70 in 10 μg HeLa (Lanes 1 and 2, observed at 71 kDa). Lane 3 - PBS. For western blotting, HRP Veriblot for IP Detection Reagent (ab131366) was used for detection (1/10 000). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

  • Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    All lanes : Anti-Hsc70 antibody [EP1531Y] (ab51052) at 1/500 dilution

    Lane 1 : Wild-type A431 whole cell lysate
    Lane 2 : HSPA8 knockout A431 whole cell lysate
    Lane 3 : HeLa whole cell lysate
    Lane 4 : MCF7 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 71 kDa
    Observed band size: 73 kDa
    why is the actual band size different from the predicted?



    Lanes 1 - 4: Merged signal (red and green). Green - ab51052 observed at 73 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

    ab51052 was shown to react with HSPA8 in A431 wild-type cells in Western blot. Loss of signal was observed when HSPA8 knockout sample was used. A431 wild-type and HSPA8 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab51052 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 500 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    All lanes : Anti-Hsc70 antibody [EP1531Y] (ab51052) at 1/5000 dilution (purified)

    Lane 1 : C6 cell lysate
    Lane 2 : PC-12 cell lysate
    Lane 3 : Mouse heart tissue lysate
    Lane 4 : Rat heart tissue lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

    Predicted band size: 71 kDa
    Observed band size: 71 kDa



    Blocking and dilution buffer: 5% NFDM/TBST.

  • Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Western blot - Anti-Hsc70 antibody [EP1531Y] (ab51052)
    All lanes : Anti-Hsc70 antibody [EP1531Y] (ab51052) at 1/5000 dilution (purified)

    Lane 1 : human fetal brain lysate
    Lane 2 : HeLa cell lysate
    Lane 3 : HEK293 cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

    Predicted band size: 71 kDa
    Observed band size: 71 kDa



    Blocking buffer: 5% NFDM/TBST
    Dilution buffer: 5% NFDM/TBST

  • Anti-Hsc70 antibody [EP1531Y] (ab51052)
    Anti-Hsc70 antibody [EP1531Y] (ab51052)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (22)

Publishing research using ab51052? Please let us know so that we can cite the reference in this datasheet.

ab51052 has been referenced in 22 publications.

  • Zheng X  et al. A circulating extracellular vesicles-based novel screening tool for colorectal cancer revealed by shotgun and data-independent acquisition mass spectrometry. J Extracell Vesicles 9:1750202 (2020). PubMed: 32363013
  • Sanwald JL  et al. The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles. Cells 9:N/A (2020). PubMed: 32560054
  • Song L  et al. KIBRA controls exosome secretion via inhibiting the proteasomal degradation of Rab27a. Nat Commun 10:1639 (2019). PubMed: 30967557
  • Abdel-Nour M  et al. The heme-regulated inhibitor is a cytosolic sensor of protein misfolding that controls innate immune signaling. Science 365:N/A (2019). PubMed: 31273097
  • A M  et al. A complex containing lysine-acetylated actin inhibits the formin INF2. Nat Cell Biol 21:592-602 (2019). PubMed: 30962575
View all Publications for this product

Customer reviews and Q&As

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1-5 of 5 Abreviews or Q&A

Immunocytochemistry/ Immunofluorescence abreview for Anti-Hsc70 antibody [EP1531Y]

 Good
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 25°C
Sample
Mouse Cell (primary cortical neurons)
Specification
primary cortical neurons
Permeabilization
Yes - 0.1% Triton/PBS
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

Submitted Jun 23 2014

Immunoprecipitation abreview for Anti-Hsc70 antibody [EP1531Y]

 Average
Abreviews
abreview image
Application
Immunoprecipitation
Immuno-precipitation step
Other - Protein G Dynabeads crosslinked to antibody
Sample
Mouse Cell lysate - whole cell (mouse embryonic fibroblasts)
Specification
mouse embryonic fibroblasts
Total protein in input
500 µg
Read More

Abcam user community

Verified customer

Submitted Jun 09 2014

Question

IHC Questionnaire
1) Abcam product code ab ab51052

2) Abcam order reference number or product batch number GR28814-4
3) Description of the problem Very faint and not clean staining

4) Sample preparation:
Species Mouse retina
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other: Fixed in 4%PFA, embedded in Yazulla, cryosectioned (8-12 micromeneter thickness)
Sample preparation
Positive control none
Negative control none
5) Fixation step No
If yes: Fixative agent and concentration
Fixation time
Fixation temperature
6) Antigen retrieval method Na-citrate pH 6.0 sub-boiling condition 20 min
7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? 0,25% Triton x-1000
Permeabilizing agent and concentration:
8) Blocking agent (eg BSA, serum…):
Concentration Serum 5%
Blocking time 45 min
Blocking temperature Room temperature
9) Endogenous peroxidases blocked? No
Endogenous biotins blocked? No
10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1/100
Diluent buffer PBS Triton X-100 0,25% BSA 1%
Incubation time Overnight, 4 degrees
11) Secondary antibody:
Species: Goat
Reacts against: Rabbit
Concentration or dilution 1/200
Diluent buffer PBS Triton X-100 0,25% BSA 1%
Incubation time 45 min
Fluorochrome or enzyme conjugate ALEXA fluorophore
12) Washing after primary and secondary antibodies:
Buffer PBS pH 7,40
Number of washes 3 x 5 min
13) Detection method Flourescence microscope
14) How many times have you run this staining? 4
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody? Concentration primary antibody, with or without antigen retrieval, changing secondary antibody

Read More
Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.
The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.
I would like to reassure you that ab51052 is tested and covered by our 6 month guarantee for use in IHC-P and mouse samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.
Reviewing this case, I would like to offer some suggestions to help optimise the results from ab51052.
1.) The bright "dirty" spots that can be seen on the image are due to precipitates of the secondary antibody. I recommend to spin down antibodies at 4C and use the supernatant to prepare the antibody solution for staining.
2.) I can also suggest to try a different antigen retrieval method. This is a step that often has to be optimized. Maybe ETDA, pH 9 will deliver a better results. Please see the information I have attached to this email.
3.) Since this protein can be in the nucleus as well as in melanosome, I recommend to add an additional permeabilization step. I can suggest to use 0.1% triton X for 10 min only.
4.) ab51052 is a monoclonal antibody and therefore only binds once per protein. Maybe an amplification is needed to enhance the signal.
I would also appreciate if you can confirm some further details:
I ma not familiar with Yazulla embedding. Is it comparable to paraffin? This antibody has not been tested for frozen sections so far.
I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More

Question

Attached are gel images in accordance with your request. Please advise. Regards,

Read More
Answer

Thank you for submitting image and clarifying the protocol. The protocol however does not explain; what dilution of primary and secondary antibody was used? The protocol say the lysates were boiled between 95-1000C I presume it is a typos error. We unfortunately are unsure why the heavy chain is giving doublet. It may be possible due to partial glycosylation or due to possible glycosylation difference in heavy chains. I can recommend repeating the experiment by optimizing the antibody dilutions or lysates amount. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

Dear Tech support,  One of our customers has ordered ab51052 and used it for IP. The customer received a band roughly in the size of 70Kd corresponding to the precipitated protein. The antibody itself  gave two bands 50kd in size instead of one corresponding to it's heavy chain (and another band at 25kd presumed light chain size).  Could you speculate why there are two bands at 50kd instead of one. Thank you for your kind help. Regards,

Read More
Answer

Thank you for contacting us. Heavy chain shows band at 50 kDa. 2 bands observed at 50 kDa does this mean you are seeing a duplet. Could you provide an image of the staining and also more information about the protocol used? I have attached the WB questionnaire. Please inform your customer to fill the questionnaire. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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