Mouse monoclonal [4A10] to TSG101 - BSA and Azide free Suitable for: Flow Cyt, WB, IHC-P Reacts with: Mouse, Human Isotype: IgG1 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. Recombinant fragment within Human TSG101 aa 150 to the C-terminus. The exact immunogen sequence used to generate this antibody is proprietary information. If additional detail on the immunogen is needed to determine the suitability of the antibody for your needs, please contact our Scientific Support team to discuss your requirements. Expressed in E.coliDatabase link: Q99816 WB: Neuro-2a, C8D30, NIH/3T3, RAW 264.7, C2C12 , HeLa, HepG2, A431, K562 and THP-1 whole cell lysate, mouse testis.ICC/IF: Amyloid peptide-treated mouse astrocytesIHC-P: human ovarian cancer tissue, human breast carcinoma tissue.Flow cyt: THP-1 cells 常规说明 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.The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q As 存放说明 Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles. 纯化说明 Purified from tissue culture supernatant by Protein G chromatography to at least 95% homogeneity as determined by SDS-PAGE. The Abpromise guarantee Abpromise™承诺保证使用ab83于以下的经测试应用 \"应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。 Flow Cyt Use at an assay dependent concentration. ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody. 1/500 - 1/3000. Detects a band of approximately 47 kDa (predicted molecular weight: 43 kDa). WB1/500 - 1/3000. Detects a band of approximately 47 kDa (predicted molecular weight: 43 kDa). IHC-P1/100 - 1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. 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. 序列相似性 Belongs to the ubiquitin-conjugating enzyme family. UEV subfamily.Contains 1 SB (steadiness box) domain.Contains 1 UEV (ubiquitin E2 variant) 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. 翻译后修饰 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. 细胞定位 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. All lanes : Anti-TSG101 antibody [4A10] - BSA and Azide free (ab83) at 1/500 dilutionLane 1 : Neuro-2a (Mouse neuroblastoma cell line) whole cell lysateLane 2 : C8D30 whole cell lysateLane 3 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysateLane 4 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysateLane 5 : C2C12 (mouse myoblast cell line) whole cell lysateLysates/proteins at 30 µg per lane.SecondaryAll lanes : HRP-conjugated anti-mouse IgG antibody at 1/500 dilutionPredicted band size: 43 kDa10% SDS PAGERunning conditions:80V for 15min then 140V for 40minBlocking:5% non-fat milk in TBST at room temperature for 60min.Washing conditions:5 ml TBST, 4 x 5minTransfer conditions:Semi-dry, 18 V, 60min (NC membrane)Exposure system:Trident plus Western HRP Substrate Immunohistochemical analysis of paraffin-embedded humanovarian cancertissuelabeling TSG101 with ab83 at 1/100 dilution. Cytoplasmic staining is observed.Antigen Retrieval: Citrate buffer, pH 6.0, 15 min. (Cuisinart Electric Pressure Cooker #EPC-1200, choose \"high pressure\"). Endogenous peroxidase blocking:3% H2O2, RT, 30min. Blocking condition:1.5% goat serum (dilute goat serum by 1xPBS), RT, 30min. Primary antibody incubation: 4 C overnight.Secondary antibody incubation:HRP Kit (Mouse IgG), 1:200, RT, 30min. Washing: PBS, 2 x 5 minutes. DAB detection. Flow Cytometry - Anti-TSG101 antibody [4A10] - BSA and Azide free (ab83)Image from Kahlert C et al., J Biol Chem. 2014;289(7):3869-75. Fig 1(C).; doi: 10.1074/jbc.C113.532267. Panc-1 and Serum Exosomes were characterized by the exosome-specific expression ofTSG101 by FACS analysis.Exosomes were attached to 4- m aldehyde/sulfate latex beadsby mixing 30 g of exosomes in a 100- l 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 thesecondary antibody. Flow Cytometry analysis of THP-1 cells labeling TSG101 with ab83 at 1/25 dilution (red).Unlabelled sample was used as a control (black).ADylight 488-conjugated secondary antibody was used for FACS analysis. All lanes : Anti-TSG101 antibody [4A10] - BSA and Azide free (ab83) at 1/500 dilutionLane 1 : Non-transfected (–) 293T whole cell extractsLane 2 : TSG101 shRNA transfected (+) 293T whole cell extractsLysates/proteins at 30 µg per lane.SecondaryAll lanes : HRP-conjugated anti-mouse IgG antibodyDeveloped using the ECL technique.Predicted band size: 43 kDa10% SDS-PAGE All lanes : Anti-TSG101 antibody [4A10] - BSA and Azide free (ab83) at 1/500 dilutionLane 1 : Mouse testis tissue extractLane 2 : Mouse pancreas tissue extractLysates/proteins at 50 µg per lane.SecondaryAll lanes : HRP-conjugated anti-mouse IgG antibodyDeveloped using the ECL technique.Predicted band size: 43 kDa Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissuelabeling TSG101 with ab83 at 1/100 dilution. Cytoplasmic staining is observed.Antigen Retrieval: Citrate buffer, pH 6.0, 15 min. All lanes : Anti-TSG101 antibody [4A10] - BSA and Azide free (ab83) at 1/500 dilutionLane 1 : K562 whole cell extractLane 2 : THP-1 whole cell extractLane 3 : HL-60 whole cell extractLysates/proteins at 30 µg per lane.SecondaryAll lanes : HRP-conjugated anti-mouse IgG antibodyDeveloped using the ECL technique.Predicted band size: 43 kDa10% SDS-PAGE All lanes : Anti-TSG101 antibody [4A10] - BSA and Azide free (ab83) at 1/500 dilutionLane 1 : NIH-3T3 whole cell lysate/extractLane 2 : JC (mouse mammary adenocarcinoma) whole cell lysate/extractLane 3 : BCL-1 whole cell lysate/extractLysates/proteins at 30 µg per lane.SecondaryAll lanes : HRP-conjugated anti-mouse IgG antibodyPredicted band size: 43 kDa10% SDS-PAGE All lanes : Anti-TSG101 antibody [4A10] - BSA and Azide free (ab83) at 1/500 dilutionLane 1 : A431 whole cell lysateLane 2 : HeLa whole cell lysateLane 3 : HepG2 whole cell lysateLane 4 : K562 whole cell lysateLane 5 : THP-1 whole cell lysateLysates/proteins at 30 µg per lane.Predicted band size: 43 kDa All lanes : Anti-TSG101 antibody [4A10] - BSA and Azide free (ab83) at 1 µg/mlLane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : A-431 whole cell lysate (ab7909)Lane 3 : Jurkat whole cell lysate (ab7899)Lane 4 : HEK-293 whole cell lysate (ab7902)Lysates/proteins at 20 µg per lane.SecondaryAll lanes : Donkey polyclonal to Mouse IgG (IRDyeTM 700DX) at 1/10000 dilutionPredicted band size: 43 kDaObserved band size: 49 kDa why is the actual band size different from the predicted? 发表研究结果有使用 ab83？请让我们知道，以便我们可以引用本数据表中的参考文章。 ab83 被引用在 178 文献中. Chen HX et al. Exosomes derived from mesenchymal stem cells repair a Parkinson\'s disease model by inducing autophagy. Cell Death Dis 11:288 (2020).PubMed: 32341347 Liu Y et al. Extracellular vesicle tetraspanin-8 level predicts distant metastasis in non-small cell lung cancer after concurrent chemoradiation. Sci Adv 6:eaaz6162 (2020).PubMed: 32195353 Castellano JJ et al. Extracellular Vesicle lincRNA-p21 Expression in Tumor-Draining Pulmonary Vein Defines Prognosis in NSCLC and Modulates Endothelial Cell Behavior. Cancers (Basel) 12:N/A (2020).PubMed: 32244977 Just J et al. Blood flow-restricted resistance exercise alters the surface profile, miRNA cargo and functional impact of circulating extracellular vesicles. Sci Rep 10:5835 (2020).PubMed: 32245988 Colangelo NW Azzam EI Extracellular vesicles originating from glioblastoma cells increase metalloproteinase release by astrocytes: the role of CD147 (EMMPRIN) and ionizing radiation. Cell Commun Signal 18:21 (2020).PubMed: 32033611 View all Publications for this product Blocking step Milk as blocking agent for 3 hour(s) and 0 minute(s) Concentration: 5% Temperature: 21 C Blocking step Milk as blocking agent for 16 hour(s) and 0 minute(s) Concentration: 5% Temperature: 4 C Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) Concentration: 5% Temperature: rt C 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 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 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 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 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 MoreThank 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 MoreThank 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. Read MorePlease note: All products are FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES For licensing inquiries, please contact partnerships@abcam.com