Isoelectric focusing in agarose under denaturating conditions

Olsson, Ingmar; Låås, Torgny

doi:10.1016/s0021-9673(00)81416-8

Cited by 25 publications

(6 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…to remove background staining. Urea/agarose isoelectric focusing Analytical isoelectric focusing in the presence of 6 Murea was as described by Olsson & Laas [16]. Gels were prefocused for 15 min at 1500 V to allow the formation Multiple forms of serum A-esterase activity of the pH 4-7 gradient, before the addition of 20 ,ug of protein dissolved in 10 mM-Tris/HCl buffer, pH 8.2, containing 7 M-urea, and focusing for 1 h in a watercooled LKB 2117 Multiphor II electrophoresis unit.…”

Section: Analytical Polyacrylamide-gel Electrophoresismentioning

confidence: 99%

“…Gels were prefocused for 15 min at 1500 V to allow the formation Multiple forms of serum A-esterase activity of the pH 4-7 gradient, before the addition of 20 ,ug of protein dissolved in 10 mM-Tris/HCl buffer, pH 8.2, containing 7 M-urea, and focusing for 1 h in a watercooled LKB 2117 Multiphor II electrophoresis unit. After the focusing, proteins were fixed and stained as described by Olsson & Laas [16].…”

Section: Analytical Polyacrylamide-gel Electrophoresismentioning

confidence: 99%

See 1 more Smart Citation

Multiple forms of sheep serum A-esterase activity associated with the high-density lipoprotein

Mackness

Walker

1988

Biochemical Journal

View full text Add to dashboard Cite

Five lipoproteins of sheep serum expressing A-esterase activity, but with differing activities towards four organophosphate substrates, were separated by a combination of gel filtration and ion-exchange chromatography. Each had an Mr of approx. 360,000 and contained a major peptide of Mr 28,000-30,000 that appeared to be present as several isoforms on urea/agarose isoelectric focusing. In every case this peptide split into a number of bands on urea/agarose isoelectric focusing. The bands appear to represent isoforms of the peptide, and four lipoproteins yielded characteristic patterns of bands. This peptide resembles the apolipoprotein A-I of human serum, and available evidence suggests that this is the protein that expresses A-esterase activity. Evidence is presented for the existence of different species of high-density lipoprotein HDL2 particles containing different complements of peptide isoforms and expressing contrasting substrate specificities towards organophosphates.

show abstract

Section: Analytical Polyacrylamide-gel Electrophoresismentioning

confidence: 99%

Section: Analytical Polyacrylamide-gel Electrophoresismentioning

confidence: 99%

Multiple forms of sheep serum A-esterase activity associated with the high-density lipoprotein

Mackness

Walker

1988

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…A PH 3-11 agarose (0.5%) isoelectric focusing gel (Pharmacia, Uppsala, Sweden) containing 0.5% Triton X-100 was prepared on a piece of gel bond according to Olsson and Laas (1981). It was pre-focused at 500 V for 30 min.…”

Section: Lsoelectric Focusingmentioning

confidence: 99%

“…Raji cell extract (0.02 ml) was applied together with PI markers. Isoelectric focusing was performed at 1,OOO V for 1.5 hr (Olsson and Laas, 1981). One portion of the gel was fixed and stained by Coomassie brilliant blue and the other portion was blotted onto a nitrocellulose sheet according to Schibeci et al (1986).…”

Section: Lsoelectric Focusingmentioning

confidence: 99%

Characterization of a human B‐lymphocyte carcinoma cross‐reacting antigen (BLCa) in B lymphocytes identified by two murine monoclonal antibodies

Yip

Chan

1987

Intl Journal of Cancer

View full text Add to dashboard Cite

The murine monoclonal antibody (MAb) MA6 is selectively reactive against a large variety of human B lymphocytes including those in early stages of B-cell differentiation such as committed progenitors of B lymphocytes, pre-B lymphocytes, Burkitt lymphoma cells, and those at later stages of differentiation such as peripheral blood B lymphocytes and myeloma cells. The major antigen identified by this antibody on such B lymphocytes (BLCa) is a 55-kDa glycoprotein or a group of similar glycoproteins, with the MA6-reactive determinant localized on the carbohydrate moiety. On isoelectric focusing, this antigen exhibits a degree of charge microheterogeneity, migrating as a diffused band with an average isoelectric point at pH 5.7. BLCa was also identified by another murine MAb, MA5. The antigenic determinant recognized by this antibody is also localized on the carbohydrate moiety of the molecule, but, unlike the MA6-reactive determinant, it is shared by other glycoproteins from different types of cells.

show abstract

“…Briefly, nitrocellulose transfers were incubated with radiolabeled IgG, extensively washed, and then eluted with freshly deionized 8 M urea/1% mercaptoethanol/0.05% Triton X-100/20 ,ug BSA/10 mM phosphate buffer, pH 4 at room temperature. Approximately 30,000 cpm of affinity purified antibodies were applied to ultrathin IEF agarose gels containing 8 M urea (35), 0.05% Triton X-100, as well as ampholytes 5-8 (LKB Instruments, Inc., Gaithersburg, MD), 8-9.5 (LKB Instruments, Inc.), and 2-11 (Serva Biochemicals, Westbury, NY) in a 2:1:2 ratio. After IEF the gels were either autoradiographed or subjected to SDS-PAGE in the second dimension (13) and then autoradiographed.…”

Section: Methodsmentioning

confidence: 99%

Analysis of autoantibodies to recombinant La (SS-B) peptides in systemic lupus erythematosus and primary Sjogren's syndrome.

Bini¹,

Chu²,

Okolo³

et al. 1990

J. Clin. Invest.

View full text Add to dashboard Cite

Autoantibodies to a polymerase III transcription factor, La (SS-B), are frequently detected in the serum of patients with Sjogren's syndrome and systemic lupus erythematosus. To define the humoral immune response to this protein, we analyzed the patterns of antibody recognition toward 13 recombinant La peptides by immunoblotting and determined the heterogeneity of antibodies reactive with the immunodominant epitopes. The smallest epitopes that were strongly antigenic and recognized by > 70% of sera tested (immunodominant) were encoded by the subclones BgX and XA located in the 5' and 3' halves of the La cDNA, respectively. Conformation of the immunodominant La peptides played a major role in antibody recognition. Although greater diversity in antibody binding to carboxyl-terminal La peptides was observed, the overall pattern of peptide recognition by anti-La antibodies was similar in different diseases. The antibody responses to the immunodominant peptides were strongly correlated (r = 0.68, P < 0.001). One-and two-dimensional isoelectric focusing of affinity purified IgG anti-La peptide antibodies revealed restricted heterogeneity and oligoclonal bands (K light chains). These observations suggest that anti-La antibodies are induced and/or maintained by the self antigen and that their diversity is constrained either by mechanisms related to tolerance or by affinity maturation of the humoral immune response. (J. Clin. Invest. 1990. 85:325-333.) autoantibody -epitope mapping * La (SS-B) * systemic lupus erythematosus

show abstract

Isoelectric focusing in agarose under denaturating conditions

Cited by 25 publications

References 7 publications

Multiple forms of sheep serum A-esterase activity associated with the high-density lipoprotein

Multiple forms of sheep serum A-esterase activity associated with the high-density lipoprotein

Characterization of a human B‐lymphocyte carcinoma cross‐reacting antigen (BLCa) in B lymphocytes identified by two murine monoclonal antibodies

Analysis of autoantibodies to recombinant La (SS-B) peptides in systemic lupus erythematosus and primary Sjogren's syndrome.

Contact Info

Product

Resources

About