Anti cd81

Manufactured by Proteintech

Sourced in United States

Anti-CD81 is a laboratory antibody reagent used to detect and quantify the presence of CD81 protein in biological samples. CD81 is a tetraspanin protein that plays a role in various cellular processes. This antibody can be utilized in techniques such as Western blotting, flow cytometry, and immunohistochemistry to study the expression and distribution of CD81 in different cell types and tissues.

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Lab products found in correlation

Anti cd9, by Proteintech (7 mentions) Anti cd63, by Proteintech (5 mentions) Anti cd63, by Abcam (3 mentions) Anti tsg101, by Abcam (3 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions) Anti β actin, by Beyotime (2 mentions) Gel doc xr, by Bio-Rad (2 mentions) Anti vegf, by Proteintech (2 mentions) Anti hsp90, by Proteintech (2 mentions) Anti α tubulin, by Beyotime (2 mentions) Anti gapdh, by Beyotime (2 mentions) Anti calnexin, by Abcam (2 mentions) Anti tsg101, by Proteintech (2 mentions) Anti gapdh, by Proteintech (2 mentions) Anti β actin, by Cell Signaling Technology (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Chemical gel imaging system, by GE Healthcare (2 mentions) Anti β actin, by ABclonal (2 mentions) Anti hur, by Proteintech (1 mentions)

Spelling variants of this product

Anti-TM (2 citations) Anti-CD81 (66866-1-Ig, (2 citations) Anti-CD81 antibody (2 citations) Rabbit anti-CD81 (2 citations)

12 protocols using anti cd81

Western Blot, Immunohistochemistry, and Immunoprecipitation Protocols

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Proteins in cells and tissues were extracted with RIPA lysis buffer (Thermo Fisher). Serum proteins were extracted with Serum Protein Extraction Kit (Qcheng Bio, China). Western blot assays were performed according to details previously reported [23 (link)]. the immuno-complexes were detected with ECL Western Blotting Substrate (Thermo Fisher), visualized with BIO-RAD (BIO-RAD Gel Doc XR+, USA). The following antibodies were used (11000): anti-β-actin (Beyotime, AF0003); anti-α-tubulin (Beyotime, AF0001); anti-GAPDH (Beyotime, AF0006); anti-HSP90 (Proteintech, 60,318–1-Ig); anti-HUR (Proteintech, 11,910–1-AP); anti-EIF2S1 (Proteintech, 11,170–1-AP); anti-VEGF (Proteintech, 19,003–1-AP); anti-Calnexin (Abcam, ab92573); anti-CD63 (Abcam, ab134045); anti-TSG101 (Abcam, ab125011); anti-CD81 (Proteintech, 66,866–1-Ig); anti-STUB1 (Proteintech, 55,430–1-AP).
IHC, IF and IP was performed as previously described [24 (link)]. IHC was performed with antibodies against HUR and VEGF (Proteintech, 1:200). IF was performed with antibody against CD31 (Proteintech, 11,265–1-AP, 1:200). The images were scanned by Pannoramic SCAN (3DHistech, Hungary). IP was performed with anti-HSP90 antibody (Proteintech, 1:200) and appropriate control IgG (Merck Millipore), and the immunoprecipitate was then collected by centrifugation and analysed by SDS-PAGE.

Xie M., Yu T., Jing X., Ma L., Fan Y., Yang F., Ma P., Jiang H., Wu X., Shu Y, & Xu T. (2020). Exosomal circSHKBP1 promotes gastric cancer progression via regulating the miR-582-3p/HUR/VEGF axis and suppressing HSP90 degradation. Molecular Cancer, 19, 112.

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Western Blot Analysis of Signaling Proteins

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Proteins were subjected to SDS-PAGE on polyacrylamide gels (8–10%) and transferred onto a PVDF membrane. After blocking with 5% non-fat milk in TBS containing 0.1% Tween-20, the membrane was incubated at 4 °C overnight with one of the following primary antibodies: anti-p-NF-κB P65, anti-NF-κB P65, anti-p-mTOR, anti-p-ERK, anti-ERK, anti-PDCD4, anti-GNA12 (Affinity, USA); anti-mTOR, anti-GAPDH, anti-TSG101, anti-CD63, anti-CD81, anti-Bax (Proteintech, USA); anti-Bcl-2 (Abclonal, China). Subsequently, the peroxidase-conjugated AffiniPure goat anti-rabbit or mouse IgG (Proteintech, USA) was added. Bound antibody was visualized via ECL plus TM Western blotting system detection kit (Amersham, USA).

Bai X., Qi Z., Zhu M., Lu Z., Zhao X., Zhang L, & Song G. (2022). The effect of lncRNA MIR155HG-modified MSCs and exosome delivery to synergistically attenuate vein graft intimal hyperplasia. Stem Cell Research & Therapy, 13, 512.

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Exosome Protein Profiling in Renal Tissues

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Homogenized renal tissues, cells, and exosome lysates were separated via 8% or 12% SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Jaffrey, NH, USA), and probed with the following antibodies: anti-CD9 (#20597-1-AP, 1:500; Proteintech), anti-CD63 (#ab216130, 1:500; Abcam), anti-CD81 (#18250-1-AP, 1:500; Proteintech), anti-VEGF (#19003-1-AP; 1:1000, Proteintech), anti-HIF-1α (#AF1009, 1:1000; Affinity), anti-SIRT1 (#13161-1-AP, 1:500; Proteintech), anti-eNOS (#AF0096; 1:1000, Affinity), anti-p-eNOS (#AF3247, 1:1000; Affinity) and anti-β-actin (#4970, 1:1000; Cell Signaling Technology).

Chen L., Wang Y., Li S., Zuo B., Zhang X., Wang F, & Sun D. (2020). Exosomes derived from GDNF-modified human adipose mesenchymal stem cells ameliorate peritubular capillary loss in tubulointerstitial fibrosis by activating the SIRT1/eNOS signaling pathway. Theranostics, 10(20), 9425-9442.

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Western Blot Analysis of Proteins

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Proteins extracted from cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, U.S.A.), which were then blocked using 5% dried skimmed milk powder. Blots were incubated with primary antibodies at 4°C overnight and then incubated with the secondary antibody for 1 h at room temperature. Specific proteins were visualized using the Odyssey Infrared Imaging System (Li-COR, Lincoln, NE, U.S.A.) according to the manufacturer’s instructions. The primary antibodies and their dilutions used included anti-CD81 (1: 500) (Proteintech, Wuhan, China) (Cat. No. 18250-1-AP), anti-GAPDH (1: 100000) (Proteintech, Wuhan, China) (Cat. No. 60004-1-Ig).

Zhang N., Zuo L., Zheng H., Li G, & Hu X. (2018). Increased Expression of CD81 in Breast Cancer Tissue is Associated with Reduced Patient Prognosis and Increased Cell Migration and Proliferation in MDA-MB-231 and MDA-MB-435S Human Breast Cancer Cell Lines In Vitro. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 5739-5747.

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Exosomal Protein Expression Analysis

Cited 1 time

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Western blotting was performed as described previously [9] (link). Primary antibodies against CD9, CD63, CD81, HSP90, FGFBP1, SIPA1, THBS1, TGFBI, COL6A1, RPL10, SLC2A3 (GLUT3), MYO1D, RBP1, SMOC2, GLG1, and CEMIP (Protein-Tech Group, Rosemont, IL, USA) were used.
Western blotting was performed to verify the expression of exosome marker proteins and selected exoDEPs in SW620 and SW480 exosomes. Equivalent amounts of total protein (20 μg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the antibodies were diluted as follows: anti-CD9 (1:1000), anti-CD63 (1:1000), anti-CD81 (1:2000), anti-HSP90 (1:200), anti-FGFBP1 (1:500), anti-SIPA1 (1:2000), anti-THBS1 (1:500), anti-TGFBI (1:200), anti-COL6A1 (1:200), anti-RPL10 (1:1000), anti-GLUT3 (1:500), anti-MYO1D (1:200), anti-RBP1 (1:500), anti-SMOC2 (1:500), anti-GLG1 (1:1000), and anti-CEMIP (1:1000) (Protein-Tech Group, Rosemont, IL, USA). Proteins were detected using an enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Dallas, TX, USA). Western blot signals were quantified using FluorChem E (Protein Simple, San Jose, CA, USA). All analyses were performed using western blots in at least two biological replicates.

Liu X., Li N., Zhang C., Wu X., Zhang S., Dong G, & Liu G. (2022). Identification of metastasis-associated exoDEPs in colorectal cancer using label-free proteomics. Translational Oncology, 19, 101389.

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Comprehensive Protein Extraction and Analysis

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Total cellular protein was extracted using RIPA lysis buffer with PMSF and phosphatase inhibitor solution. The concentration of the proteins was determined by a BCA protein quantitative kit. The protein samples (40 μg/lane) were separated by SDS-PAGE and transferred onto activated PVDF membranes (Invitrogen, CA). After blocking with 5% skim milk, the membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies and dilutions were as follows: anti-ARID2 (1:1000, Cell Signaling Technology, USA), anti-CD63, anti-CD9, anti-p-ERCC5, anti-CD81, anti-p-NF-KB, and anti-NF-KB (1:500, Proteintech, USA). After washing with TBST, the membranes were incubated with the appropriate secondary antibodies. The protein bands were visualized with an ECL kit (Thermo, USA).

Li H., Chi X., Li R., Ouyang J, & Chen Y. (2019). HIV-1-infected cell-derived exosomes promote the growth and progression of cervical cancer. International Journal of Biological Sciences, 15(11), 2438-2447.

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Western Blot Analysis of Exosomal Proteins

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RIPA lysate (Pierce) was added to the tissues/cells to obtain the protein that was mixed with the loading buffer (Life Technologies) at a ratio of 3:1, and boiled at 100°C for 8‐10 min. Protein samples were added to the gel well and the target protein was separated by electrophoresis. The protein was transferred to a PVDF membrane (Millipore), and non‐specific antigens blocked with 5% skim milk. The membranes were incubated with corresponding primary antibodies: anti‐CD9 (1:500; Proteintech), anti‐CD63 (1:500; Proteintech), anti‐CD81 (1:500; Proteintech), anti‐NEDD8 (1:500; CST), anti‐cullin 1 (1:500; Abcam), anti‐IκB (1:500; CST), anti‐NF‐κB (1:500; Abclonal), anti‐P‐NF‐κB (1:500; Abclonal), anti‐PCNA (1:500; Abcam), and anti‐β‐actin (1:500; SAB) overnight at 4°C. After washing with 1× TBS/T buffer, the membranes were incubated with the secondary antibody for 30 min at 37°C. Pictures were taken with a chemical gel imaging system (GE).

Wang G., Yuan J., Cai X., Xu Z., Wang J., Ocansey D.K., Yan Y., Qian H., Zhang X., Xu W, & Mao F. (2020). HucMSC‐exosomes carrying miR‐326 inhibit neddylation to relieve inflammatory bowel disease in mice. Clinical and Translational Medicine, 10(2), e113.

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Extracellular Vesicle Characterization Protocols

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The following antibodies and reagents were used for this study: anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA, cat#a3854), anti-ALIX (Abcam, Cambridge, UK, cat#ab186429), anti-CD63 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, cat#sc-5275), anti-CD81 (Proteintech, Wuhan, China, cat#66866-1-lg), anti-CD9 (Proteintech, cat#60232-1-lg), anti-Tsg101 (Abcam, cat#ab133586), anti-Exo70 (Abcam, cat#ab118792), anti-F-actin (Cytoskeleton, Inc., Denver, CO, USA; cat#PHDR1), anti-PD-L1 (Cell Signaling, Danvers, MA, USA, cat# 13684), Endosidin2 (ES2) (Cayman Chemical, Ann Arbor, MI, USA, cat#21888) and PKH67 (Sigma-Aldrich, cat#MINI67).

Xiang J., Zheng B., Zhao L., He Y., Lou F., Li R., Fu M., Huang X., Zhang W., Hong X., Xiao L, & Hu T. (2024). Exo70 Promotes the Invasion of Pancreatic Cancer Cells via the Regulation of Exosomes. Cancers, 16(2), 336.

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Exosome Protein Extraction and Western Blot

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A total exosome RNA and protein isolation kit (Thermo Fisher) was utilized to extract protein from exosomes. Radioimmunoprecipitation (RIPA) lysis buffer was employed to lyse the culture cells, and the total protein was then collected. Protein samples were denatured and subjected to SDS‐PAGE electrophoresis, followed by electrophoresis on a nitrocellulose membrane (Boster) and immersed in 5% skimmed milk. The membranes were probed with primary antibodies, including anti‐E‐cad (#14472, Cell Signaling), anti‐N‐cad (#14215, Cell Signaling), anti‐vimentin (#3390, Cell Signaling), and anti‐PSAT1 (#67619‐1‐Ig, Proteintech), anti‐CD63 (#67605‐1‐Ig, Proteintech), anti‐CD81 (#66866‐1‐Ig, Proteintech), anti‐TSG101 (#67381‐1‐Ig, Proteintech), and anti‐β‐actin (#3700, Cell Signaling) antibodies. After incubation with a secondary antibody, the bands were visualized using chemiluminescence (Thermo Fisher).

Peng X., Zhao L., Yao L., Dong J., Wu W, & Luo T. (2023). Exosomal ERBB2IP contributes to tumor growth via elevating PSAT1 expression in non‐small cell lung carcinoma. Thoracic Cancer, 14(19), 1812-1823.

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Western Blot Analysis of Exosomal Markers

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RIPA lysate (Thermo Fisher Scientific, MA, USA) was applied to colon tissues and cells and protein concentration was measured by the BCA method. Protein samples (200 μg) were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was transferred to a PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% skim milk to reduce non-specific antigens exposure. The PVDF membranes were incubated with the primary antibodies: anti-CD9 (1:500; Proteintech Group), anti-CD63 (1:500; Abcam, Cambridge, UK), anti-CD81 (1:500; Proteintech Group), anti-HSP70 (1:500; Abcam), anti-Alix (1:500; Cell Signaling Technology), anti-Calnexin (1:500; Abcam), anti-NLRP3 (1:1000; Novus), anti-ASC (1:1000; Novus), anti-Caspase-1 p45 (1:1000; Proteintech Group), anti-Caspase-1 p20 (1:200; Santa Cruz Biotechnology), anti-IL-1β (1:500; Cell Signaling Technology), anti-IL-18 (1:200; Wanleibio, Shenyang, China), anti-GSDMD (1:500; Cell Signaling Technology), and anti-β-actin (1:10000; Abclonal, Boston, MA, USA) at 4 °C overnight, followed by the HRP-conjugated secondary antibodies for 30 min at 37 °C. A chemical gel imaging system (GE Healthcare Life sciences China, Beijing, China) was used to visualize protein bands and generate images.

Cai X., Zhang Z.Y., Yuan J.T., Ocansey D.K., Tu Q., Zhang X., Qian H., Xu W.R., Qiu W, & Mao F. (2021). hucMSC-derived exosomes attenuate colitis by regulating macrophage pyroptosis via the miR-378a-5p/NLRP3 axis. Stem Cell Research & Therapy, 12, 416.

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