Sodium dodecyl sulfate (sds)

The sulfhydryl groups were determined using the Ellman modified method described by Chan and Wasserman (1993 ). Briefly, 1 ml of 40 mM DTNB solution (5,5′dithiobis(2-nitrobenzoic acid); Sigma Aldrich, USA) was added to the sample (86 mM Tris (Serva, Germany), 90 mM glycine (Serva, Germany), 4 mM EDTA (Sigma Aldrich, USA), 8 M urea (Avantor, Poland), 0.5% SDS (sodium dodecyl sulfate, Serva, Germany), 0.2 M Tris HCl (Trizma base, Sigma Aldrich, USA)), pH 8. Next, 200 μl of the samples were added to 1 ml of DTNB solution. Samples were incubated at room temperature for 30 min. Cysteine was used (Sigma Aldrich, USA) as a standard and the absorbance was measured by a spectrophotometer (Perkin Elmer, Lambda 25, Biocompare, USA) at a wavelength of λ = 412 nm. The concentration of –SH groups was measured from a calibration curve based on a Cysteine solution in PBS. The concentration of the thiol groups was presented as micromolar per milligram of whole protein in the supernatant of spleen.

All cell culture reagents, RPMI, fetal calf serum (FCS), phosphate buffered saline (PBS), penicillin, and streptomycin, were purchased from PAA Laboratories, Colbe, Germany. Acetonitrile (ACN) was purchased from Promochem, Wesel, Germany. CHAPS was obtained from Applichem, Darmstadt, Germany. Urea, thioUrea, dithiothreitol (DTT), trypsin, trifluoroacetic acid (TFA), sodium carbonate, ammonium bicarbonate, 6-MP, 6-TG, and DMSO were obtained from Sigma-Aldrich, Steinheim, Germany. Ampholytes, protein assay kits, and immobilized pH gradient strips (IPG strips) were obtained from Bio-Rad, Munich, Germany. Protease and phosphatase inhibitor cocktails were purchased from Roche, Mannheim, Germany. Glycerin, potassium ferricyanide, and sodium thiosulfate were purchased from Merck, Darmstadt, Germany. Formic acid was from BASF, Ludwigshafen, Germany. Bromophenol blue and Trizma base were from Carl Roth, Karlsruhe, Germany. Sodium dodecyl sulfate (SDS) was purchased from Serva, Heidelberg, Germany.

NanoPro 1000 reagents were obtained from ProteinSimple (Santa Clara, USA). Dry milk and urea was obtained from Roth (Karlsruhe). 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was from Merck (Darmstadt). DL-Dithiothreitol (DTT), iodoacetamide (IAA) and thiourea was obtained from Sigma (Steinheim, Germany). IPG buffer was from Amersham Biosciences (Freiburg). Sodium dodecyl sulfate was bought from Serva (Heidelberg). Tris pure was obtained from Applichem (Darmstadt). Polyvinylidene fluoride membranes were purchased from Millipore Corporation (Bedford). IPG strips were from GE Healthcare (Munich). The monoclonal mouse anti-human serpinA1 antibody was bought from R&D Systems (MAB1268) (Minneapolis).

Reagents and their sources were as follows: phosphate buffer saline (PBS) (PAA Laboratories, Cölbe, Germany), sodium carbonate, ammonium bicarbonate, thiourea, dithiothreitol (DTT), urea, trypsin, trifluoroacetic acid‎ (TFA) (Sigma-Aldrich), CHAPS (AppliChem, Darmstadt, Germany), Acetonitril (ACN) (Promochem, Wesel, Germany), Immobilized pH gradient strips (IPG strips), ampholytes, protein assay kit (Bio-Rad, Munich, Germany), protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany), sodium dodecyl sulfate (SDS) (Serva, Heidelberg, Germany), potassium ferricynaide, Glycerin, sodium thiosulfate (Merck, Darmstadt, Germany), formic acid (BASF, Ludwigshafen, Germany), bromophenol blue (Carl Roth, Karlsruhe, Germany).

GOx from Aspergillus niger was purchased from Sigma-Aldrich (St. Louis, MO, USA). Peroxidase from horseradish (HRP) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from AppliChem (Darmstadt, Germany). Sodium metaperiodate (NaIO₄) was purchased from VWR Chemicals (Leuven, Belgium). Zinc acetate dihydrate (Zn(O₂CCH₃)₂·2H₂O) and D-glucose monohydrate were purchased from Lachner (Neratovice, Czech Republic), while HmIM was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium dodecyl sulfate (SDS) was purchased from Serva (Heilderberg, Germany). All reagents were of analytical grade and were used as purchased. All solutions were prepared with distilled water.

Cell culture media Dulbecco's Modified Eagle's medium (DMEM), fetal calf serum (FCS), phosphate buffer saline (PBS), penicillin and streptomycin were purchased from PAA Laboratories, Pasching, Austria. Fluorescein isothiocyanate-conjugated dextran (FITC-dextran), 4 kDa (FD4), Trypsin, MPA, and DMSO were purchased from Sigma-Aldrich, Steiheim, Germany. Protease and phosphatase inhibitor cocktails were purchased from Roche, Mannheim, Germany. Bromophenol blue was obtained from Carl Roth, Karlsruhe, Germany. Sodium dodecyl sulfate (SDS) was obtained from Serva, Heidelberg, Germany. Glycerin, potassium ferricynaide, and sodium thiosulfate were purchased from Merck, Darmstadt, Germany. Formic acid was purchased from BASF, Ludwigshafen, Germany. Magnesium chloride (MgCl₂), M-MLV RT enzyme, and 5X PCR buffer were from Invitrogen, Karlsruhe, Germany. Deoxynucleotide triphosphates (dNTPs) were from Roche, Mannheim, Germany and PCR primers were synthesized by Eurofins, Ebersberg, Germany. Ribonuclease (RNAase) inhibitor was obtained from Promega, Mannheim, Germany. The cell lysis buffer (10X) was obtained from Cell Signaling Technology, Danvers, MA, USA. All other chemicals used in this work were from the highest available purity from commercial sources unless otherwise stated.