| Isotype | IgG1 (F)/IgG1 (PE) |
| Product Type | Bi-Testª Reagents (FITC/RPE) |
| Units | 50 Tests |
| Host | Mouse |
| Species reactivity | Human |
| Application | Flow Cytometry |
BackgroundIdentification of human cytotoxic/suppressor T cells expressing the 32 and 43,000 M.W. surface antigen. CD38 antigen is an integral membrane glycoprotein, M.W. 45 kD. Anti-human CD28 binds the 44kDa MW cell surface protein on the surface of most T cells. CD28 acts as the ligand for theB7/BB-1 molecule on the surface of activated B cells. B7/BB-1 co-stimulates T cells through CD28, along with CD2 and CD3. CD28 antigen is a disulfide-linked homodimeric glycoprotein. The CD28 antigen is present on approximately 60%-80% T lymphocytes (95% of CD4 and 50% of CD8lymphocytes). CD28 regulates the expression of cytokines by T cells, not only IL-2, but also IL-1 alpha and CSF-1, usually synthesized by accessory cells. CD28 functions as a cell adhesion molecule (CAM) for certain T cell subsets.
Synonyms: CD8 FITC - CD28 PE
Source
Immunogen: CD8=Derived from the hybridization of mouse NS-1 myeloma cells with spleen cells from BALB/c mice immunized with human perherial blood T lymphocytes.CD28=Derived from the hybridization of mouse Sp2/O-Ag14 myeloma cells with spleen cells of BALB/c mice immunized with the HPB-ALL T-cell line.
Product
Product Form: Bi-Test (FITC/RPE) Reagent
Formulation: Provided as solution in phosphate buffered saline with 0.08% sodium azide and 0.2% carrier protein
Purification Method: Protein A/G Chromatography
Concentration: Titered for flow cytometry
ApplicationsPBMC: Add 10 µl of MAB/10^6 PBMC in 100 µl PBS. Mix gently and incubate for 15 minutes at 2ºto 8ºC. Wash twice with PBS and analyze or fix with 0.5% v/v of paraformaldehyde in PBS and analyze. WHOLE BLOOD: Add 10µl of MAB/100 µl of whole blood. Mix gently and incubate for 15 minutes at room temperature (20º C). Lyse the whole blood. Wash once with PBS and analyze or fix with 0.5% v/v of paraformaldehyde in PBS and analyze.See instrument manufacturer’s instructions for Lysed WholeBlood and Immunofluorescence analysis with a flow cytometer or microscope.Storage: Antibodies are supplied in PBS, 0.08% sodium azide and 0.2% protein carrier for FITC andPE. Antibodies should be stored at 4-8oC. Mabs should not be frozen.Reagents are stable for the period shown on the vial label when stored properly.
Functional Analysis: Flow Cytometry Staining
StorageProduct should be stored at 4-8ºC. DO NOT FREEZE
Product Stability: Reagents are stable for the period shown on the vial label when stored properly
Shipping Conditions: Room Temperature
CautionThis product is intended FOR RESEARCH USE ONLY, and FOR TESTS IN VITRO, not for use in diagnostic or therapeutic procedures involving humans or animals. It may contain hazardous ingredients. Please refer to the Safety Data Sheets (SDS) for additional information and proper handling procedures. Dispose product remainders according to local regulations.This datasheet is as accurate as reasonably achievable, but Nordic-MUbio accepts no liability for any inaccuracies or omissions in this information.
References1. Evolutionary Conservation of Surface Molecules that Distinguish T Lymphocyte Helper/Inducer and T Cytotoxic/Suppressor Subpopulations in Mouse and Man. Ledbetter,J.A., Evans,R.L., Lipinski,M., Cunningham-Rundles,C., Good,R.A. and Herzenberg,L.A., J. Exp. Med. 153,310, 1981. 2. Circulating Antigen-Specific Suppressor T Cells in a Healthy Woman: Mechanism of Action and Isolation with a Monoclonal Antibody.Engleman,E.G.,Benike,C.J.,and Evans,R.L., Clin. Res. 29, 365a 1981. 3. Induction of Immunoglobulin Secreting Cells in the Allogeneic Mixed Leukocyte Reaction: Regulation by Helper and Suppressor Lymphocyte Subsets in Man.Kotzin,B.L., Benike,C.J. and Engleman,E.G., J. Imm. 127,931,1981. 4. CD4 and CD8 molecules can physically associate with the same T-cell receptor. Gallagher,P.F., Fazekas de St. Groth,B., Miller, J.F.A.P., Proc. Nat. Acad. Sci. 1989,86:10044. 5. Immunofluorescence Measurement in a Flow Cytometer using Low-Power Helium Neon Laser Excitation.Shapiro, H.M, Glazer, A.N., Christenson, L., Williams, J.M., and Strom, T. B. Cytometry 4,276, 1983. 6. Comparison of Helium Neon and Dye lasers for Excitation of Allophycocyanin.Loken, M.R., Kiej, J.F. and Kelly, K.,A.Cytometry 8, 96, 1987. 7. CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes. Turka LA; Ledbetter JA; Lee K; June CH; Thompson CB J Immunol 1990 Mar ;144(5):1646-538. CD28 ligation in T-cell activation: evidence for two signal transduction pathways. Ledbetter JA; Imboden JB; Schieven GL; Grosmaire LS; Rabinovitch PS; Lindsten T; Thompson CB; June CH Blood 1990 Apr ;75(7):1531-99. T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-1. Linsley PS; Clark EA; Ledbetter JA Proc Natl Acad Sci U S A 1990 Ju;87(13):5031-510. Role of the CD28 receptor in T-cell activation. June CH; Ledbetter JA; Linsley PS; Thompson CB Immunol Today 1990 Ju;11(6):211-611. Differences in surface phenotype and mechanism of action between alloantigen-specific CD8+ cytotoxic and suppressor T cell clones. Koide J; Engleman EG J Immunol 1990 Jan ;144(1):32-4012. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. Linsley PS; Brady W; Grosmaire L; Aruffo A; Damle NK; Ledbetter JA J Exp Med 1991 Mar ;173(3):721-30 13 13. The CD28 ligand B7/BB1 provides costimulatory signal for alloactivation of CD4+ T cells. Koulova L; Clark EA; Shu G; Dupont B J Exp Med 1991 Mar ;173(3):759-62
Protein Reference(s)
Database Name: UniProt
Accession number: P01732, P10747
Safety Datasheet(s) for this product:NM_Sodium Azide/wp-content/uploads/SDS/Antibody SDS with Sodium Azide Noridic-MUbio.pdf
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其次,察看次目的蛋白的存在形式,有没有多聚体形式及变构形式;
最后,查看多家抗体公司的DATA,看看别人的WB做出来的条带的位置。
根据你说的,特异识别多个组织中的同样大小的条带,我觉得很可能就是你的目的蛋白。
一般来说多克隆的阳性率高一些,但出现假阳性的比例也高一些。
1取组织
(1) 灌注固定:暴露心脏,将注射针头插入左心室,剪开右心耳,快速灌注PBS,待右心耳处流出澄清液体,换4%多聚甲醛灌注到大鼠四肢强直为止,快速取脑和肾。
(2) 固定:将组织置于4%多聚甲醛中固定15小时
(3) 脱水:将组织放入30% 蔗糖溶液中直至下沉
(4) 切片:脑组织冰冻切片20微米,37度烘干2h以上;
(5)将切片放置-80度保存,随用随取。
2免疫单标记
(1)做抗原修复(0.4g柠檬酸、3g柠檬酸钠、1000ml水配制成溶液,放到水浴箱中90~94℃保存15min),冷却后, 用1*PBS反复洗标本10min,吸去PBS,同时向标本加渗透液(配0.4%trition液,然后将BSA加入到配好的trition液中,配成1%浓度)100ul,常温下湿盒反应1h。
(2) 吸去渗透液,同时向标本加block液(5%封闭用正常山羊血清)100ul,常温下湿盒反应5h。
(3) 吸去block液,同时向标本加入1:50稀释的羊抗鼠一抗100ul,37度湿盒反应过夜。
(4)吸去一抗,并回收,用1*PBS洗液反复洗15min。
(5)快速从冰箱中取出驴抗羊的二抗,并立即用锡纸包裹,注入到一个新的以锡纸包裹的tube管中并用PBS以1:200稀释。
(6)进入暗室,吸去PBS,同时加入二抗100ul,37度湿盒反应2h,封片。
(7)放置到荧光显微镜下观察。
又由于自然存在的抗原大都存在多个抗原表位,会刺激机体产生多种针对同一抗原的不同抗原表位相应的不同抗体.

