Overview

  • Product name
    Anti-TSG101 antibody [4A10]
    See all TSG101 primary antibodies
  • Description
    Mouse monoclonal [4A10] to TSG101
  • Host species
    Mouse
  • Specificity
    This antibody recognizes the TSG-101 protein, the product of a recently identified tumor susceptibility gene the inactivation of which in mouse fibroblasts results in cell transformation and the ability of those cells to form tumors in nude mice.
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, IP, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Drosophila melanogaster
  • Immunogen

    Fusion protein containing amino acids 167-374 of the TSG101 protein expressed in E. coli.

  • Positive control
    • WB: Neuro-2a, C8D30, NIH/3T3, RAW 264.7 , C2C12 , HeLa, HepG2, A431, K562 and THP-1 whole cell lysate. ICC/IF: Amyloid peptide-treated mouse astrocytes IHC-P: Colon carcinoma tissue.
  • General notes

    This product was changed from ascites to tissue culture supernatant on 12th February 2018. Please note that the dilutions may need to be adjusted accordingly. If you have any questions, please do not hesitate to contact our scientific support team.

Properties

Applications

Our Abpromise guarantee covers the use of ab83 in the following tested applications.

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

Application Abreviews Notes
Flow Cyt Use at an assay dependent concentration. PubMed: 23045692

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

 

ICC/IF Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB 1/100 - 1/1000. Detects a band of approximately 47 kDa (predicted molecular weight: 43 kDa).
IHC-P 1/100. Do not perform antigen retrieval.

Target

  • Function
    Component of the ESCRT-I complex, a regulator of vesicular trafficking process. Binds to ubiquitinated cargo proteins and is required for the sorting of endocytic ubiquitinated cargos into multivesicular bodies (MVBs). Mediates the association between the ESCRT-0 and ESCRT-I complex. Required for completion of cytokinesis; the function requires CEP55. May be involved in cell growth and differentiation. Acts as a negative growth regulator. Involved in the budding of many viruses through an interaction with viral proteins that contain a late-budding motif P-[ST]-A-P. This interaction is essential for viral particle budding of numerous retroviruses.
  • Tissue specificity
    Heart, brain, placenta, lung, liver, skeletal, kidney and pancreas.
  • Sequence similarities
    Belongs to the ubiquitin-conjugating enzyme family. UEV subfamily.
    Contains 1 SB (steadiness box) domain.
    Contains 1 UEV (ubiquitin E2 variant) domain.
  • Domain
    The UEV domain is required for the interaction of the complex with ubiquitin. It also mediates the interaction with PTAP/PSAP motifs of HIV-1 P6 protein and human spumaretrovirus Gag protein.
    The coiled coil domain may interact with stathmin.
    The UEV domain binds ubiquitin and P-[ST]-A-P peptide motif independently.
  • Post-translational
    modifications
    Monoubiquitinated at multiple sites by LRSAM1 and by MGRN1. Ubiquitination inactivates it, possibly by regulating its shuttling between an active membrane-bound protein and an inactive soluble form. Ubiquitination by MGRN1 requires the presence of UBE2D1.
  • Cellular localization
    Cytoplasm. Membrane. Nucleus. Late endosome membrane. Mainly cytoplasmic. Membrane-associated when active and soluble when inactive. Depending on the stage of the cell cycle, detected in the nucleus. Colocalized with CEP55 in the midbody during cytokinesis.
  • Information by UniProt
  • Database links
  • Alternative names
    • ESCRT I complex subunit TSG101 antibody
    • ESCRT-I complex subunit TSG101 antibody
    • TS101_HUMAN antibody
    • TSG 10 antibody
    • TSG 101 antibody
    • TSG10 antibody
    • Tsg101 antibody
    • Tumor susceptibility 101 antibody
    • Tumor susceptibility gene 10 antibody
    • Tumor susceptibility gene 101 antibody
    • Tumor susceptibility gene 101 protein antibody
    • Tumor susceptibility protein antibody
    • Tumor susceptibility protein isoform 3 antibody
    • VPS 23 antibody
    • VPS23 antibody
    see all

Images

  • All lanes : Anti-TSG101 antibody [4A10] (ab83) at 1/500 dilution

    Lane 1 : Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate.
    Lane 2 : C8D30 whole cell lysate.
    Lane 3 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate.
    Lane 4 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate.
    Lane 5 : C2C12 (mouse myoblast cell line) whole cell lysate.

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : HRP-conjugated anti-mouse IgG antibody

    Predicted band size: 43 kDa

  • Immunohistochemical analysis of paraffin-embedded colon carcinoma tissue labeling TSG101 with ab83 at 1/100 dilution.

  • Immunofluorescence analysis of amyloid peptide-treated mouse astrocytes, staining TSG101 with ab83.

    Astrocytes were fixed with paraformaldehyde and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. Cells were incubated with primary antibody and a fluorescent-conjugated anti-mouse IgG was used as the secondary antibody.
  • All lanes : Anti-TSG101 antibody [4A10] (ab83) at 1/500 dilution

    Lane 1 : A431 whole cell lysate
    Lane 2 : HeLa whole cell lysate
    Lane 3 : HepG2 whole cell lysate
    Lane 4 : K562 whole cell lysate
    Lane 5 : THP-1 whole cell lysate

    Lysates/proteins at 30 µg per lane.

    Predicted band size: 43 kDa

  • Panc-1 and Serum Exosomes were characterized by the exosome-specific expression of TSG101 by FACS analysis.

    Exosomes were attached to 4-μm aldehyde/sulfate latex beads by mixing ∼30 μg of exosomes in a 100-μl volume of beads for 2 hours at room temperature. This suspension was diluted to 1 ml with PBS, and the reaction was stopped with 100 mm glycine and 2% BSA in PBS. Exosome-bound beads were washed in PBS/1% BSA, blocked with 10% BSA, and stained for FACS with anti-TSG101 (1:400, ab83). An Alexa Fluor® 488 conjugated anti-mouse IgG was used as the secondary antibody.

  • All lanes : Anti-TSG101 antibody [4A10] (ab83) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : A431 whole cell lysate (ab7909)
    Lane 3 : Jurkat whole cell lysate (ab7899)
    Lane 4 : HEK293 whole cell lysate (ab7902)

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Donkey polyclonal to Mouse IgG (IRDyeTM 700DX) at 1/10000 dilution

    Predicted band size: 43 kDa
    Observed band size: 49 kDa
    why is the actual band size different from the predicted?

References

This product has been referenced in:
  • Mleczko J  et al. Extracellular Vesicles from Hypoxic Adipocytes and Obese Subjects Reduce Insulin-Stimulated Glucose Uptake. Mol Nutr Food Res 62:N/A (2018). Read more (PubMed: 29292863) »
  • Santasusagna S  et al. Proteomic Analysis of Liquid Biopsy from Tumor-Draining Vein Indicates that High Expression of Exosomal ECM1 Is Associated with Relapse in Stage I-III Colon Cancer. Transl Oncol 11:715-721 (2018). WB . Read more (PubMed: 29660691) »
See all 104 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (Hela)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Specification
Hela
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Mar 20 2018

Answer

Thank you for contacting us.

You can use any protease inhibitor, most common are Leupeptin and PMSF. These protease inhibitors should be dissolved in lysis buffer.

People tend to use protease inhibitors cocktails which have many enzymes in it. These can bought from Sigma, Roche or other vendors. Please use these in lysis buffer as per manufacturer's instructions.

Chemically denaturation generally does not affect primary structure, but it does cause degradation of the complex three-dimensional arrangements of the proteins structures e.g. secondary, tertiary, and quaternary. Most protein functions result from the three-dimensional arrangements of the proteins, so denaturation of such structures generally results in a loss of protein function. In western blot proteins gest denatured at high temperature with degradation of secondary structures. The primary structuresare still intact for antibodies to bind.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

Thank very much for filling the questionnaire and for sending the image.

I have reviewed the protocol now and would like to point out few things that may be the cause of multiple bands;

- Protease inhibitors; You have mentioned that no protease inhibitors were used but these are necessary to use in lysis buffer. I would suggest using these and redoing the lysis.

- In our own lab we heat the samples in loading buffer at 100C for 10 minutes. I would suggest heating the samples for 5-10 minutes.

- We have observed a single band in Hela, Jurkat, HEK293 and A431 cell lines; these cell lines are easily available so lysates of these can be used as positive control.

- Try loading 20ug protein.

- Trying a no primary control will help to check the non specificity of secondary antibody.

There are two isoforms of this protein one is 44kDa and other is 32 kDa. Please click the link http://www.uniprot.org/uniprot/Q99816 for more info. In 32 kDa isoform amino acid 15-112 is missing. The antibody recognize the epitope between 167-374 so it is more likely that the antibody will detect both the isoform but the isoform should be expressed in the samples. We are however not sure if the isoform is expressed in monocytes.

The Abcam anti TSG1 antibody has been used in following publication http://www.nature.com/bjc/journal/v92/n2/full/6602316a.html. In Fig 1 duplet has been observed. This publication may provide further insight into you r research.

I hope these suggestions will help to improve the results. If however the results do not improve please contact me I will be happy to assist further.

Read More

Answer

Thanks you for your email.

There could be many reasons for this extra band. It could be an Isoform; TSG101 have 2 isoforms one is 31kDa nd other is 44 kDa. Two bands could be observed however it will depend on the lysates being used. http://www.uniprot.org/uniprot/Q99816

It could be non specific band, which can go away with simple protocol troubleshooting e.g.
- Using 70-80% confluent cells
- Using Fresh protease inhibitors
- Using fresh samples

We have used Hela, Jurkat, A431 and kidney cells while testing this antibody and we have observed no extra band which could means that the isoform is not expressed in cell line. I would recommend using the lysates of same as positive control.

I can provide further help however would need to know more about the protocol. I have attached a questionnaire with my email, please fill in and send it to me asap.

I hope these suggestions will help to improve the results.

Read More

Answer

Thank you for contacting Abcam.

Forthese antibodies I would recommend using reducing conditions and also initially using a 1/1000 primary antibody dilution, although you may to alter this concentration for subsequent western blots.

If there is anything else I can help you with, please let me know.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Human HeLa)
Loading amount
30 µg
Specification
Human HeLa
Gel Running Conditions
Reduced Denaturing (12.5% gel)
Blocking step
Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Nov 25 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - other (brain, HEK cell lysate)
Loading amount
50 µg
Specification
brain, HEK cell lysate
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: rt°C

Dr. Sriharsha Kantamneni

Verified customer

Submitted Sep 26 2007

Answer

The datasheet was correct and the customer should have received 100ul, I apologise for this problem and have arranged a free of charge vial to be sent to you. Many thanks for your help with this matter,

Read More

Answer

Thank you for your email. I contacted the originator of this antibody who was able to provide me with the following information. "Concerning IHC staining, you may want to refer to the following publication for a detailed protocol: Zhu et at, Oncogene 22:3742 (2003). 1. The authors in the previously-referenced paper were using paraffin-embedded tissues (this particular group was looking at human tissues. I do not have an IHC reference for rat or mouse). 2. No antigen retrieval was necessary. 3. Following thorough washing in 0.015 m TBS (pH 7.6) and blocking with 20% normal rabbit serum, the antibody, at 1:100, was applied for 1 hr. 4. Sites of antIgen-antibody binding were detected using biotinylated rabbit anti-mouse Ig followed by peroxidase conjugated streptavidin–biotin complex cDNA. Unfortunately, I do not have any information concerning the use of this antibody with frozen tissue sections." If you need further assistance, please contact us again.

Read More

Question

I'm adding two coplaint protocols for different applications: I Abcam Troubleshooting form ?Immunohistochemistry- 1. Please describe the problem (high background, no staining etc). weak but overall, unspecific staining of the whole cell. No specific labelling, as it was seen before with previously purchased batch. Upon redistribution of protein (overexpression of GFP-tagged mutant construct), no redistribution of ab83 labelling, i.e. no co-localization of protein and antibody in IF. In Immuno-EM: overall staining of all cellular compartments in ultrathin cryosections 2. On what material are you testing the antibody in IHC? Cultured cell lines, primary cells ? Species? human ? Cell line? HelaP4, MT4, primary macrophages. ? Tissue? 3. How did you fix the samples? ? 3% Paraformaldehyde in PBS for fluorescence ? Other: Glutaraldehyde+PFA or PFA only for immuno-EM 4. Did you apply antigen retrieval step? NO ? Enzymatic method ? Heat mediated technique ? Other 5. How did you block the unspecific binding sites? 5% FCS in PBS/20mM glycine OR 5% goat serum in PBS/20mM glycine OR 1%fish skin gelatine/0.8% BSA in PBS/20mM glycine 6. Primary antibody ? Specification: ab83 Mouse monoclonal (4A10) AB to Tsg101, cross reacts with human and mouse. Lot 506 ? At what dilution(s) have you tested this antibody? 1:10- 1:300 ? Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? Several attempts: 20-60 min incubation. Wash (PBS/glycine of PBS alone) 3-6 steps, 2-4 each, depending on number of wash steps. 7. Secondary antibody ? What secondary antibody are you using? IgGs: Donkey anti-mouse Cy3 Or donkey anti-mouse FITC for IF. For Immuno-EM: rabbit-anti-mouse IgG + ProteinA Gold ? Specification (in which species was it raised against)? See above ? At what dilution(s) have you tested this antibody? As established for the secondary abs in the lab (1:400 Cy3, 1:250 for FITC, 1:75 for rabbit-anti-mouse). ? Incubation, wash steps?. As for primary AB ? Do you know whether the problems you are experiencing come from the secondary?. Yes, I know. It's not the secondaries, since they work fine for all other applications. Negative controls (i.e. in this case incubation with secondary AB alone) are truly negative ? What detection method are you using? IF: fluorescence microscopy, wide-field and confocal. Electron microcopy: ProteinA Gold + uranyl acetate contrasting. 8. Background staining ? Please provide an image of your staining. See attachment "Tsg.ppt". 9. Which detection system did you use? 10. Did you apply positive and negative controls along with the samples? IF Negative control: secondary only (since Tsg101 is an endogenous protein). Additional control: redistribution of Tsg101 to internal aberrant endosomal structures (dn Vps4B overexpression). This leads to no difference in ab83 staining 11. Optimization attempts ? How many times have you tried the IHC? 3 ? Do you obtain the same results every time? yes ? What steps have you altered? AB dilution, blocking solution, incubation time II Abcam Troubleshooting form ?Immunohistochemistry- 1. Please describe the problem (high background, no staining etc). weak but overall, unspecific staining of the whole cell. No specific labelling, as it was seen before with previously purchased batch. Upon redistribution of protein (overexpression of GFP-tagged mutant construct), no redistribution of ab83 labelling, i.e. no co-localization of protein and antibody in IF. In Immuno-EM: overall staining of all cellular compartments in ultrathin cryosections 2. On what material are you testing the antibody in IHC? Cultured cell lines, primary cells ? Species? human ? Cell line? HelaP4, MT4, primary macrophages. ? Tissue? 3. How did you fix the samples? ? 3% Paraformaldehyde in PBS for fluorescence ? Other: Glutaraldehyde+PFA or PFA only for immuno-EM 4. Did you apply antigen retrieval step? NO ? Enzymatic method ? Heat mediated technique ? Other 5. How did you block the unspecific binding sites? 5% FCS in PBS/20mM glycine OR 5% goat serum in PBS/20mM glycine OR 1%fish skin gelatine/0.8% BSA in PBS/20mM glycine 6. Primary antibody ? Specification: ab83 Mouse monoclonal (4A10) AB to Tsg101, cross reacts with human and mouse. Lot 506 ? At what dilution(s) have you tested this antibody? 1:10- 1:300 ? Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? Several attempts: 20-60 min incubation. Wash (PBS/glycine of PBS alone) 3-6 steps, 2-4 each, depending on number of wash steps. 7. Secondary antibody ? What secondary antibody are you using? IgGs: Donkey anti-mouse Cy3 Or donkey anti-mouse FITC for IF. For Immuno-EM: rabbit-anti-mouse IgG + ProteinA Gold ? Specification (in which species was it raised against)? See above ? At what dilution(s) have you tested this antibody? As established for the secondary abs in the lab (1:400 Cy3, 1:250 for FITC, 1:75 for rabbit-anti-mouse). ? Incubation, wash steps?. As for primary AB ? Do you know whether the problems you are experiencing come from the secondary?. Yes, I know. It's not the secondaries, since they work fine for all other applications. Negative controls (i.e. in this case incubation with secondary AB alone) are truly negative ? What detection method are you using? IF: fluorescence microscopy, wide-field and confocal. Electron microcopy: ProteinA Gold + uranyl acetate contrasting. 8. Background staining ? Please provide an image of your staining. See attachment "Tsg.ppt". 9. Which detection system did you use? 10. Did you apply positive and negative controls along with the samples? IF Negative control: secondary only (since Tsg101 is an endogenous protein). Additional control: redistribution of Tsg101 to internal aberrant endosomal structures (dn Vps4B overexpression). This leads to no difference in ab83 staining 11. Optimization attempts ? How many times have you tried the IHC? 3

Read More
Answer

Thank you for the details that you have provided and I'm sorry to hear that your customer is experiencing difficulty with this antibody. You mentioned that a previously purchased batch worked well; do you have that batch number? Were the protocols and samples the same for both batches? Thank you, and we look forward to hearing from you.

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1-10 of 11 Abreviews or Q&A

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