Overview

  • Product name

  • Description

    Rabbit polyclonal to TSG101
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, ICC/IF, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human TSG101 aa 350 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
    Database link: Q99816
    (Peptide available as ab30870)

  • Positive control

    • This antibody gave a positive signal in the following whole cell lysates: HeLa (Human epithelial carcinoma cell line) A431 (Human epithelial carcinoma cell line) Jurkat (Human T cell lymphoblast-like cell line) HEK293 (Human embryonic kidney cell line) NIH 3T3 (Mouse embryonic fibroblast cell line) PC12 (Rat adrenal pheochromocytoma cell line) This antibody also gave a positive signal in human placenta tissue sections.

Properties

Applications

Our Abpromise guarantee covers the use of ab30871 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
WB Use a concentration of 1 µg/ml. Detects a band of approximately 48,49 kDa (predicted molecular weight: 44 kDa).Can be blocked with TSG101 peptide (ab30870).

The doublet seen in Western blot has been described in PMID:11427703 and may be due to internal initiation at Met10.

ICC/IF Use a concentration of 5 µg/ml.
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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 (ab30871) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 4 : HEK293 Human embryonic kidney cell line Whole Cell Lysate
    Lane 5 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 6 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
    Lane 7 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate
    Lane 8 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 44 kDa
    Observed band size: 49 kDa
    why is the actual band size different from the predicted?

  • IHC image of ab30871 staining in human placenta formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab30871, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  • ab30871 stained in HeLa cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab30871 at 5µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

  • All lanes : Anti-TSG101 antibody (ab30871) at 1 µg/ml

    Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 2 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 44 kDa
    Observed band size: 48,49 kDa why is the actual band size different from the predicted?
    Additional bands at: 29 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 90 seconds


    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab30871 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

    The doublet seen in Western blot has been described in PMID:11427703 and may be due to internal initiation at Met10.

References

This product has been referenced in:

  • Langevin SM  et al. Balancing yield, purity and practicality: a modified differential ultracentrifugation protocol for efficient isolation of small extracellular vesicles from human serum. RNA Biol 16:5-12 (2019). Read more (PubMed: 30604646) »
  • Jimenez L  et al. Quantitative Proteomic Analysis of Small and Large Extracellular Vesicles (EVs) Reveals Enrichment of Adhesion Proteins in Small EVs. J Proteome Res 18:947-959 (2019). Read more (PubMed: 30608700) »
See all 47 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Answer



I think it would be helpful to have a positive control sample in this instance. The online data for ab30871 shows a western blot where tissue extracts of mouse and rat liver and kidney exhibit detectable levels of TSG101. I would recommend running a western blot with one of these or another positive control sample.

If one of these sample types is not available, please note we do sell tissue extracts ready to load on a SDS- PAGE gel, such as:

https://www.abcam.com/Liver-Mouse-Whole-Cell-Lysate-normal-tissue-ab29301.html

Read More

Answer

Thank you very much for your interest in ab30871.

To our knowledge, ab30871 has not been tested in african green monkey and we do not know yet if this antibody will work with Vero cells. I can confirm ab30871 is tested and guaranteed to work in ICC/IF ( with human, mouse and rat).

Therefore, I can offer a discount off a future purchase if you buy ab30871 now, test it in african green monkey and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free primary antibody.

If you are interested in this offer, please follow these steps:

1. Reply to this e-mail to let me know that you would like to proceed and test ab30871 in african green monkey. I will then send a discount code. This code must be issued before purchasing ab30871 so please wait for my reply before ordering.

2. Purchase ab30871 either by phone, fax, or online (www.abcam.com).

3. Test it in african green monkey.

4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: https://www.abcam.com/abreviews.

5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any primary antibody ordered and the discount code is valid for 4 months after issue.

We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab30871 turns out to be unsuitable for african green monkey, you will still receive the discount on your next purchase after your Abreview has been submitted.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

Read More

Answer

Thank you for contacting Abcam regarding ab30871. I am sorry that you have been experiencing difficulties with this antibody in WB. I have reviewed the protocol information you have provided and would like to make some suggestions that may improve your results. Regarding your samples - how are the lysates prepared? What buffer and protease inhibitors do you include? Can you provide an image of your results? Are you detecting a band around the expected molecular weight or are all bands non-specific and faint? For the primary antibody incubation, it is important to include a blocking reagent in the diluent to help reduce non-specific bands. I recommend diluting the antibody in 5% milk in TBST. Additionally, it is not necessary to include milk in the washes and you can perform washes with TBST alone. TBS is recommended instead of PBS here to help reduce background as well, but is not absolutely necessary to change. I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

Read More
Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa)
Loading amount
20000 cells
Specification
HeLa
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

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Verified customer

Submitted Oct 03 2008

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