CHAPTER 048. ACIDOSIS AND ALKALOSIS (PART 9) PDF

Chapter 048. Acidosis and Alkalosis (Part 9) Approach to the Patient: Hyperchloremic Metabolic Acidoses In diarrhea, stools contain a higher [HCO3–] and decomposed HCO3– than plasma so that metabolic acidosis develops along with volume depletion. Instead of an acid urine pH (as antici[r]

4. Apply the oxidised mucin to a Sephadex G25 column as for the biotinylation of protein-labeled mucin (see Note 6) and collect fractions.5. Detect mucin containing fractions mucin using the slot-blot assay with the periodic acidSchiff’s stain (see Chapter 4), and pool these fractions (approx 5 × 1[r]

UK) and 0.25 mg/mL of hyaluronidase (type 1; Sigma, Poole, UK).d. 3T3 Conditioned medium: DMEM is supplemented with 10% FBS, 2 mM glutamine,100 IU/mL of penicillin, 100 µg/mL of streptomycin, and put onto 24-h postconfluent3T3 cell layers for 24 h. After conditioning, the medium is filtered through[r]

GI TISSUE1. Use the submerged technique for tissue culture (Chapter 18). Place the tissue segments (oneper tube) immediately after excision in the appropriate EMEM to deplete the compound tobe used as label. Incubate for 30 min in 100 µL per tube to deplete this compound.2. Add 100 µCi (3700 kBq) of[r]

Amino Acid Analysis of Mucins 113113From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ10Amino Acid Analysis of MucinsJun X. Yan and Nicolle H. Packer1. IntroductionAmino acid analysis is a commonly used t[r]

seeNote 19)1. Prepare 0.8% agarose gels, according to standard procedures (see Chapter 19).2. Analyze the homogenates and the immunoprecipitated mucins on the agarose gels.3. Place the agarose gel on a pre-wetted piece of 3MM paper, and dry the gel immediatelyon a gel dryer.4. Expose the dried gel t[r]

4. 3% stacking/4% running gels for SDS-PAGE, electrophoresis apparatus (mini Protean IIsystem; Bio-Rad, Richmond CA), and Laemmli electrophoresis buffers.5. PhosphorImager (Molecular Dynamics, Sunnyvale, CA), with ImageQuant software (or equiv-alent apparatus) to quantify the amount of radioactivity[r]

amino acids (see Chapter 19).2. Homogenize the samples and isolate the radiolabeled mucin precursor of interest by im-munoprecipitation using specific antipolypeptide antibodies (see Chapter 20).3. Analyze the immunoprecipitated mucin precursors on 4% SDS-PAGE using reducingsample buffer.4. Identify[r]

ers are advised to refer to Chapters 1 and 2 (2,3) of this volume for the preparation ofsecreted and membrane-associated mucins, respectively, and to Chapter 7 (4) for adiscussion of methods for mucin separation.46 McGuckin and ThorntonSelection of a technique to detect mucins should be influenced b[r]

background in the proliferation assay where the DCs are used as antigen presenting cells.2. From 20 mL of blood there is normally enough PBMCs to put up three to five 33-mm dishes.3. This dislodges the lymphocytes that can be collected and used as effector cells in theproliferation assay.4. The mono[r]

7. CarboPac MA-1 column (4 × 250 mm) (Dionex).8. CarboPac MA-1 guard (4 × 50 mm) (Dionex).9. 18 M Ω deionized water (Milli-Q Plus System, Millipore, Bedford, MA).10. NaOH 50% solution with less than 0.1% sodium carbonate (Baker, Deventer, The Netherlands).11. Anhydrous sodium acetate (Merck).[r]

into Sf-9 cells by cationic liposomes. Within the cells, transfer vector DNA and viralDNAs recombine, incorporating the gene of interest into the viral genome. Dependingon the transfer vectors different protocols can be used to make recombinant virus. Twoprotocols are given next.3.3.1. Using[r]

reaction (PCR) formattable polymorphisms that are in linkage disequilibrium with theVNTR alleles, which would allow analysis of more samples and would use less DNA. Aprotocol for such a polymorphism within MUC1 is presented here (see Note 9).2. Materials (see Notes 10 and 11)1. Puregene kit f[r]

®cellulose ester membranes, 500 Da molecular weight (MW) cut-off (FisherScientific, Fair Lawn, NJ).4. BCA Protein assay kit (Pierce, Rockford, IL).From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ430 McNa[r]

of the mucus gel will be compromized and disease may result. The proportion of poly-meric mucins present in mucus gels has been shown to be an indicator of gel strength(9) and evidence exists for enhanced mucolysis by proteinases and/or inferior mucinpolymerisation and consequent impairment o[r]

tide; to MUC1 variant peptides; to MUC2, MUC3, MUC4 VNTR peptides; and tomouse muc1.From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ370 Xing et al.2. Materials1. Cell culture medium: Dulbecco’s modified[r]

3.2. Restriction Enzyme Digestion (see Notes 2 and 3)3.2.1. Restriction Enzyme Digestion of Plugs1. For restriction enzymes, choose enzymes according to the sites they recognize:a. (G+C) rich sites included in CpG islands: NotI, AscI (Group I); BssHII, SacI, KspI(Group II);b. those independant of th[r]

7. Dissolve the sample in 5 mL of 0.1 M pyridine acetate, pH 5.0, apply to a column of Bio-Gel P4 (200–400 mesh, 150 × 2 cm) (Bio-Rad), and elute in the same buffer.8. Collect 5-mL of fractions and measure radioactivity. Pool the major radioactive peak(Neu5Acα2-6GalNAc-[3H]-ol) (see Note 3).406 Corf[r]

erties. Individual mucin oligosaccharide chains may contain no acidic groups, one ormore sialic acid groups, one or more sulfate groups, or mixtures of sialic acid and sulfategroups (4,5). The histochemical description of mucin as neutral, sialo-, and sulfomucindepends on whether the mixture of olig[r]

7. Water bath (42°C) for autoradiography.8. Light-tight darkroom.2.2. Fixation and Embedding of Tissue in Paraffin1. 4% Paraformaldehyde in 0.1 M phosphate buffer, pH 7.4.2. 0.1 M phosphate buffer, pH 7.4.3. 100, 95, 70, and 50% ETOH.Fig. 1. Micrographs demonstrating two methods of disclosure of oli[r]