Hrp conjugated anti rabbit igg secondary antibody

Streptavidin-coated paramagnetic beads (100 μl, 1 μm diameter; Life Technologies) were saturated with 20 μg of enzymatically monobiotinylated DBLMSP or a Cd4 tag-alone control, isolated with a magnet, and washed three times with PBS before incubating with 1 ml of filtered human serum (Sigma) for one h at 4 °C. Beads were washed four times with 1 ml of PBS and eluted with 200 μl of 1% SDS. 20 μl were resolved by SDS-PAGE under reducing conditions and stained with SYPRO Orange (Sigma), and the gel image captured on a Typhoon 9400 phosphorimaging device (GE Healthcare).
Anti-human IgM agarose beads (Sigma) were incubated with long term parasite culture supernatants from the IT4 var1 and var13 strains grown in the presence of human serum or control culture medium without parasite for 1 week at 37 °C. After five washes in PBS, the beads were resuspended in loading buffer in the presence or absence of DTT, and eluates were blotted onto nitrocellulose membranes (Amersham Biosciences Protran) followed by blocking in PBS, 0.1% Tween 20, 5% nonfat milk powder) and incubated for 1 h with a rabbit anti-full-length DBLMSP antibody at a 1:100 dilution. After further washes, the membrane was incubated with an anti-rabbit HRP-conjugated IgG secondary antibody (1:1000; Sigma) and developed using 3′,3′-diaminobenzidine (DAKO) according to the manufacturer's instructions.

Antibodies against ERK1/2, phospho-ERK1/2, Akt, phospho-Akt, c-Jun N-terminal kinases (JNK), phospho-JNK, p38, phospho-p38, COX-2, 5-LOX, glyceraldehydes-3-phosphate dehydrogenase (GAPDH), β-actin, lamin b, and Alexa Fluor 594 conjugated goat anti-rabbit IgG antibodies were obtained from Cell Signaling Technology (Boston, MA, USA). Anti-NF-κB and anti-mouse horseradish peroxidase- (HRP-) conjugated IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Dihydroethidium (DHE) was purchased from molecular probes (Eugene, OR, USA). Dexamethasone was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-rabbit HRP-conjugated IgG secondary antibody and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated. Gallic acid-l-leucine (GAL) conjugate was synthesized from gallic acid and methyl-l-leucine hydrochloride by following the previous method as described [15 (link)]. The structure of GAL conjugate was confirmed by NMR technique whereas the purity of the product was determined by HPLC analysis, suggesting the purity of up to 98%.

PACA was synthesized and characterized by NMR and MS as previously described [22 (link), 23 (link)]. Cell culture medium, fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against iNOS, cyclooxygenase-2 (COX-2) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and PPAR-γ was purchased from Cell Signaling Technology (Boston, MA, USA). The antibodies against Arg1, ED2 and CD163 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against ED1 and CD68 were purchased from Abcam (Cambridge, Massachusetts, UK). Anti-mouse CD80 FITC was obtained from BD Biosciences (San Diego, CA, USA). Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody were purchased from Life Technologies (Carlsbad, CA, USA). Anti-rabbit HRP-conjugated IgG secondary antibody and other chemicals including GW9662, a specific PPAR-γ inhibitor, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

The mycelial proteome of R. solani was resolved in a 2-DE gel and electrophoretically transferred onto a Hybond-C membrane (GE Healthcare) with a blotting buffer (39 mM glycine, 48 mM Tris base, 20 % methanol, and 0.037 % SDS) using a semidry blotting apparatus (TE77; Amersham Pharmacia Biotech). The electrotransfer was run for 60 min at a current of 56 mA, 25 V. The membrane was temporarily stained with Ponceau S (Sigma-Aldrich, USA) to ensure the protein transfer from gel to Hybond-C membrane. The membrane was incubated for 15 min in Ponceau S staining solution with gentle agitation. Finally the membrane was rinsed in distilled water for two washes of 5 min each until the background is clean. Then the membrane was blocked overnight in 10 ml blocking buffer [5 % nonfat milk (Merck, Germany) in 1 × TBST]. Next day, the membrane was washed with three changes of TBST for 2 min each time and further incubated with mASAL (20 μg) for 2 h at 37 °C. Finally, the blot was incubated using a primary anti-mASAL polyclonal antibody (1:8000) and an anti-rabbit IgG HRP-conjugated secondary antibody (1:20,000, Sigma-Aldrich, USA). Membranes incubated without mASAL served as negative controls (data not shown).

Crude fractions of cell-associated polysaccharides were obtained from overnight cultures in LB essentially as described by Hitchcock and Brown [61 (link)] and Valiente [12 (link)]. Polysaccharides were separated by SDS-PAGE [62 (link)] in discontinuous gels (4% stacking gel, 10% separating gel), transferred to a PVDF membrane (Bio-Rad) [63 (link)] and subjected to immunoblot analysis. The membranes were stained with a rabbit anti-inactivated YJ016 cells serum especially enriched in antibodies against capsular antigens (anti-capsular serum) [64 (link)]. The serum was diluted 1:3000 and membranes were developed following incubation with anti-rabbit IgG HRP-conjugated secondary antibody diluted 1:10000 (Sigma), using Immobilon Western Chemiluminescent HRP Substrate (Millipore).