3. The smallest single DNA fragment that can be detected with the MUC3 TR probe SIB124 is an Swa I fragment of approx 200 kb (by pulsed field gel electrophoresis). When digested with Pst I or Pvu II, SIB124 recognises two distinct sets of very large polymorphic bands. Each set shows independent a[r]
1. Introduction Northern blot analysis has historically been one of the most common methods used to provide information on the number, length, and relative abundance of mRNAs expressed by a single gene. This technique also generates a record of the total mRNA content expressed by a cell cul[r]
An alternative to the radioactive methods is the use of biotinylated mucin sub- strates tagged through either the peptide or carbohydrate moieties of the molecule (6 , 7) . Such assays have been described for protein substrates and adapted for use with microtiter plates (8) . They require s[r]
12. Rentrop, M., Knapp, B., Winter, H., and Schweizer, J. (1986) Aminoalkylsilane-treated slides as support for in situ hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment condition. Histochem. J. 18, 271–276. 13. Briand, J. P., Muller, S., and Van Re[r]
9. Gipson, I. K. and Inatomi, T. (1999) Cellular origin of mucins of the ocular surface. in Advances in Experimental Medicine and Biology (Sullivan, D. A., ed.), Plenum, New York, pp. 221–228. 10. Gipson, I. K., Ho, S. B., Spurr-Michaud, S. J., Tisdale, A. S., Zhan, Q., Torlakovic, E.,[r]
Zouitina, S., Pigny, P. Degand, P., Aubert, J. P., and Porchet, N. (1995) Characterization of the human mucin gene MUC5AC : a consensus cysteine-rich domain for 11p15 mucin genes? Biochem. J. 305, 211–219. 16. Dufossé, J., Porchet, N., Audié, J. P., Guyonnet-Dupérat, V., Laine, A., Van Seuningen,[r]
8. It is essential to have isotonic conditions, i.e., 300 mosmolar to shield the negative charge on the mucins otherwise anomalously high viscosities will result. 9. It is vital to include controls without enzyme in the mucolysis experiments. Crude mucus samples may contain endogenous pro[r]
3. Methods 3.1. Preparation of mRNA 1. Total RNA is isolated from cultured cells or human mucosae as described in Chapter 25. In spite of the fact that the purification yield of poly A + mucin fraction is very poor, the purification step of polyA + RNA from total RNA cannot be omitted pri[r]
TRANG 12 BROCKHAUSEN TABLE 1 MUCIN GLYCOSYLTRANSFERASES, THEIR SUBSTRATES AND PRODUCTS, AND HPLC CONDITIONS FOR PRODUCT ISOLATION HPLC Path Enzyme Substrate Product column % AN a Polypep[r]
3. Methods 3.1. Cell Culture in the Presence of GalNAc α -O-benzyl 3.1.1. Conditions of Use of GalNAc α -O-benzyl GalNAc α - O -benzyl is directly dissolved in the culture medium for 2 h at room temperature with continuous stirring. Then, the medium is sterilized by filtration. GalNAc α[r]
In this Chapter, we focus on the identification of each of the known MUC-type mucin precursors by immunoprecipitation using antipeptide antibodies. Moreover, a number of biochemical and cell biological assays will be described which establish the presence in the RER of each alleged MUC-type mucin pr[r]
sodium acetate, pH 4.0); coffee bean α -galactosidase (200 m M Tris-HCl, pH 6.5); bovine testes (100 m M citrate phosphate, pH 4.0). 11. In addition to the colorimetric assays given in this chapter, additional sensitivity can be attained by the use of fluorimetric assays with 4-methyl umbellifer[r]
on a gel dryer. 4. Expose the dried gel to X-ray film or to a PhosphorImager plate ( see Note 18 ). 4. Notes 1. The methods for metabolic labeling and immunoprecipitation have been optimized for the use on gastrointestinal cell lines or tissue samples, particularly for each of the follo[r]
infected with a MUC1 recombinant baculovirus, produce fully glycosylated, full-size (no deletion of TRs) molecules that are expressed on the cell surface (6) . Moreover, under specific starvation growth conditions that we determined empirically, Sf-9 cells can also produce underglycosylate[r]
8. Horseradish peroxidase-labeled rabbit antihuman IgG and IgM, both from Dako A/S, Glostrup, Denmark. Dilute the conjugate 1:10,000 in phosphate/salt buffer containing 0.5% BSA. 9. Substrate solution containing 0.06 mg/mL of tetramethylbenzidine (TMB) diluted in 0.1 M sodium acetate/citric[r]
3. Transfer the marrow into a 20-mL universal. Allow any fragments of bone or muscle to fall to the bottom of the tube under gravity and the transfer the supernatant to a fresh universal. 4. Count cells and resuspend at 3.33 × 10 5 /mL in IMDM ( see Subheading 2., item 1e ) plus 5%FCS and mouse GM-[r]
In addition to the MHC-unrestricted CTLs, some MUC1-specific MHC-restricted CTLs have also been identified. Patients who are HLA-A11 and HLA-A3 have CTLs that recognize a nine amino acid peptide from the tandem repeat region bound to these alleles (6) . Another peptide has been reported that binds H[r]
T.L đi, đứng, bị, lẫy theo cữ và cịn hơi nhanh hơn so với các trẻ khác. Được 2 tháng tuổi cháu đã biết lẫy, 9 tháng tuổi cháu đã biết đi tốt và biết nĩi khoảng 5 từ. Gọi “T.L ơi” cháu biết “ dạ” rất rõ ràng. Hỏi cháu cĩ ăn đồ gì đĩ khơng cháu biết nĩi “ khơng ” hoặc “ cĩ”. Khi xem phim[r]
Methods outlined for the isolation of bacteria from the rectal mucosa are essentially destructive, and primarily provide details of the types and numbers of different species that take part in this process. They do not contribute information concerning the mul- ticellular organization of biof[r]
2. Wash wells 24 times with PBS. 3. Block for 2 h at room temperature (or overnight at 4 ° C) by adding 200 µ L of 1% BSA/ PBS to each well. 4. After blocking, wash wells twice with PBS/Tween and incubate with 100 µ L of 5 µ g/mL biotinylated wheat germ agglutinin (WGA) for 45 min at room tem[r]