Blotting from PhastGel media after horizontal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

Jägersten, Christina; Edström, Annica; Olsson, Birgitta; Jacobson, Gunilla

doi:10.1002/elps.1150091007

Cited by 37 publications

(9 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Gradient gels (8-25% and 4-15% polyacrylamide) were stained with Coomassie blue and silver nitrate for proteins, and toluidine blue (1 "/i in 2O' >1. methanol) for polysaccharides. Semi-dry electrophoretic transfer to nitrocellulose after SDS PAGE of the intact HSPG and the HSPG core protein was performed [31]. prior to Western blotting.…”

Section: Methodsmentioning

confidence: 99%

Purification of Amyloid‐Associated Heparan Sulphate Proteoglycans and Galactosaminoglycan Free Chains from Human Tissues

Stenstad¹,

Magnus²,

Husby³

et al. 1993

Scand J Immunol

View full text Add to dashboard Cite

Basement membrane-associated heparan sulphate proteoglycans have been demonstrated immunohistochemically in organs from patients afflicted with various types of amyloidosis. In a recent report, we were able to isolate and partly characterize a basement membrane-associated heparin sulphate proteoglycan from human hepatic amyloid. In the present study proteoglycans were extracted with guanidine from human amyloid-laden kidney, spleen and lymph nodes. All tissues extracted with guanidine contained both heparan sulphate proteoglycan (HSPG) and galactosaminoglycan (CS/DS) free chains. Tissue staining using a monoclonal antibody against basement membrane HSPG revealed the presence of HSPG in amyloid deposits in kidney and spleen. Furthermore, following SDS-PAGE of HSPG from kidney after deaminative cleavage of the HS chains, a 15-kDa and 80-kDa protein appeared, probably representing the core protein(s). In lymph node HSPG, three core proteins of 65, 30 and 25 kDa could be demonstrated on SDS-PAGE, the first reacting with the anti-basement membrane HSPG antibody when subjected to Western blotting subsequent to SDS-PAGE. By immunohistochemistry, we failed to demonstrate any staining of the renal and splenic tissue sections employing an antibody against the decorin core protein.

show abstract

Section: Methodsmentioning

confidence: 99%

Purification of Amyloid‐Associated Heparan Sulphate Proteoglycans and Galactosaminoglycan Free Chains from Human Tissues

Stenstad¹,

Magnus²,

Husby³

et al. 1993

Scand J Immunol

View full text Add to dashboard Cite

show abstract

“…After electro-phoretic transfer, the unoccupied protein binding sites were blocked by incubation with a 0.05 mol/l sodium phosphate buffer, pH 7.4, containing 0.5% Tween 20 for 30 min at room temperature. The efficiency of the blotting procedure was checked by comparing the blotted gel strips and the nonblotted by a sensitive silver staining method (25).…”

Section: Immunoblottingmentioning

confidence: 99%

“…The nitrocellulose membranes with blotted proteins were cut into strips and incubated overnight at room temperature on a shaker with patient serum, dilution 1:2 (v/v) (25). They were then washed 3 times for 10 min with 0.15 mol/l sodium chloride containing 0.5% Tween 20 followed by incubation with isotope-labelled antibody (1251 anti-IgE, 300 000 cpm/ml, Phadebas RAST isotope) for 20 h on a shaker at room temperature.…”

Section: Immunoblottingmentioning

confidence: 99%

Antigenicity and allergenicity of cow milk hydrolysates intended for infant feeding

Oldæus

Björkstén

Einarsson

et al. 1991

Pediatric Allergy Immunology

View full text Add to dashboard Cite

Allergenicity and antigenicity of various commercially available cow milk hydrolysates intended for infant feeding were analysed in 45 children with cow milk allergy. The hydrolysates included the whey hydrolysates Beba HA® (Good Start HA®) and Profylac®, and the casein hydrolysates Alimentum® and Nutramigen®. Positive skin prick tests were recorded against Beba HA in 10 of 41 tested children (24%), against Profylac® in 5/34 (15%) and in one each (2.5%) against Alimentum and Nutramigen. Double‐blind placebo‐controlled oral challenge tests were performed in 11 children with cow milk allergy using Alimentum, cow milk (positive control) and their regular well‐tolerated formula (Nutramigen or soy) used as negative control. One child reacted to Alimentum. This patient was the only one with circulating antibodies against the product, as indicated by a positive RAST. High density SDS‐PAGE electrophoresis showed that Beba HA contained a number of unresolved proteins, and non‐degraded or partially degraded whey proteins in the range of 5–20 kD. Profylac contained strongly stained protein material in the low molecular weight region 1–10 kD. No protein bands could be identified in the casein‐based hydrolysates. Residual antigenicity was tested by measuring the content of betalactoglobulin in the hydrolysates. Three of the hydrolysates contained < 0.06 μg/g dry weight, while the concentration in Beba HA was 200 μg/g dry weight. Positive RAST against Beba HA was detected in 11/45 sera (24%) compared to 7–13% against the other hydrolysates. RAST inhibition with the hydrolysates using cow milk discs was very low for all of them. Using dot immuno‐binding assay a weak IgE binding with Alimentum was detected in 4 sera, Beba HA and Profylac in each 2 sera and with Nutramigen in one. The data taken together show that all 4 tested hydrolysates retain some allergenicity. There were differences between the products, one of the whey hydrolysates being substantially more allergenic and antigenic than the other tested formulas. The casein hydrolysate Alimentum showed few reactions in vivo and in vitro in this selected group of children but one child reacted when challenged with Alimentum, indicating that there is a risk for general reactions when using any hydrolysed product in subjects allergic to cow milk.

show abstract

“…The sample (10 pl) was boiled for 5 min and 4 pl sample buffer containing bromophenyl-blue (BPB) was added. Aliquots (1 pl) were run in the Phast-system on precast gradient polyacrylamide gels, 8-25% [24]. The gels were stained with coomassie blue or transferred onto nitrocellulose membranes.…”

Section: Sds-page and Western Blottingmentioning

confidence: 99%

On the association between amyloid fibrils and glycosaminoglycans; possible interactive role of Ca2+ and amyloid P-component

Stenstad

Magnus

Syse

et al. 1993

Clinical and Experimental Immunology

View full text Add to dashboard Cite

SUMMARYWe have previously reported the specific association of glycosaminoglycans (GAG) and proteoglycans (PG) with amyloid fibrils and characterized the polysaccharides directly extracted from amyloid-laden tissues. In the present study we further elucidate the association between purified amyloid fibrils and GAG/PG with special reference to those GAG/PG associated with amyloid Pcomponent (AP) and the interactive role of Ca2+ ions. Amyloid fibrils were isolated from human hepatic AA amyloid employing water extraction with and without preceding removal of AP, an extrafibrillar protein component of all amyloids, using sodium citrate. GAG/PG co-isolated with the amyloid extracts, with and without AP, were isolated and characterized. Agarose-affinity chromatography of extracts containing AP was performed, and the GAG associated with this extrafibrillary protein were characterized as well. Several different GAG/PG populations were demonstrated in the various extracts. The abolition of calcium-dependent binding markedly influenced the amount of GAG/PG recovered in the fibril extracts, as well as the total amount of amyloid material obtained. Thus, it seems that calcium plays an important role in the association between the fibrils and the sugar moieties, and that a significant fraction of the GAG found in amyloid exhibits a Ca2+-dependent fibril-GAG interaction. No significant difference in the proportion between galactosaminoglycans and glucosamines was, however, disclosed when the two extraction protocols were compared, suggesting that no particular GAG species has a higher affinity for the fibrils themselves. Both dermatan/chondroitin sulphate and heparan sulphate identified in the present study exhibited a Ca2+-dependent interaction with AP, supporting previous findings. However, the amyloid-associated galactosaminoglycans found, especially the large PG appearing in small amounts, seemed to have a higher affinity for the extrafibrillar AP than the other GAG.

show abstract

Blotting from PhastGel media after horizontal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

Cited by 37 publications

References 7 publications

Purification of Amyloid‐Associated Heparan Sulphate Proteoglycans and Galactosaminoglycan Free Chains from Human Tissues

Purification of Amyloid‐Associated Heparan Sulphate Proteoglycans and Galactosaminoglycan Free Chains from Human Tissues

Antigenicity and allergenicity of cow milk hydrolysates intended for infant feeding

On the association between amyloid fibrils and glycosaminoglycans; possible interactive role of Ca2+ and amyloid P-component

Contact Info

Product

Resources

About