Amersham hyperfilm ecl

Radioactive Dot Blot analysis was carried out essentially as described in Fulde et al. (2011 (link)). Briefly, IgGs derived from different host species as well as different subclasses of human IgG were spotted onto a nitrocellulose membrane in a concentration series. The membrane was blocked with 5% skimmed milk at 4°C overnight. The following day, the membrane was washed five times by moderate shaking for 10 min at RT with 10 ml PBS supplemented with 0.05% Tween-20 (PBST). Hybridization with ¹²⁵I-SCM was performed for 4 h at room temperature (RT). Hybridization (binding) was detected after exposure of X-ray films (Kodak).
Non-radioactive Dot Blot analyses were performed for determination of the minimal IgG interaction sites of the SCM protein. Truncated fragments of SCM were immobilized by spotting onto nitrocellulose membranes. Blocking and washing procedures were performed as described previously (Fulde et al., 2011 (link)). HRP-conjugated rabbit anti-goat antiserum was applied as a ligand for interaction studies. Binding was detected by chemiluminescence determination after incubation of the blots with Pierce® ECL Western Blotting substrate according to the recommendations of the manufacturer. Signal development was performed by exposure of Amersham ECL-Hyperfilm (GE Healthcare) followed by an automated development with a Typon C2 developer (NDTMED-Röntgentechnik).

The amino acid sequence of Lol p 1 was verified using the UniProtKB (Lol p 1 in UniProtKB/Swiss-Prot) (P14946). SPOT synthesis technology (JPT Peptide Technologies, Germany) was used to bind covalently via C-terminus 63 overlapping synthetic peptides containing 15 amino acids to a Whatman 50 cellulose support (Whatman, UK). Peptides were overlapping by 11 amino acids and acetylated at the N-terminus to prevent degradation.
The membrane was rinsed with HPLC grade methanol (Fisher Scientific Ltd., UK) for 5 min and washed with TRIS buffered saline (TBS) + 0.05% Tween 20 (Sigma) three times. It was followed by blocking with 2% non-fat dry milk concentrate in borate buffer (KPL, UK) diluted with TBS 1/10 for 2 h at room temperature. The wash step was repeated 5 × 5 min to remove excess of blocking buffer. After washing the membrane was incubated with anti-Lol p 1 immunodominant polyclonal IgG sera (1:5,000) (Charles River Laboratories, UK) overnight at 4 °C. On the following day the membrane was washed with TBS + 0.05% Tween 20 (Sigma) five times and probed with the secondary antibody for two hours at room temperature: Goat-anti-Rabbit IgG-HRP (1:20,000) (KPL, UK). The membrane was developed using Prime ECL detection reagent (GE Healthcare, UK) and Amersham ECL Hyperfilm (GE Healthcare, UK).

To examine protein levels, N. benthamiana was infiltrated with the relevant Agrobacterium transformants, with leaf tissue sampled 48 h postinfiltration and immediately frozen in liquid nitrogen. Protein extraction was performed by heating ground tissue samples at 95°C for 10 min in 2x SDS gel-loading buffer (100 nm Tris-HCl, pH 6.8, 0.2% [w/v] bromophenol blue, 20% [v/v] glycerol, and 4% [w/v] SDS), followed by centrifugation at 16,000g for 5 min. Proteins were separated on 4% to 12% Bis-Tris PAGE gels with MES buffer, using an X-blot Mini Cell (Thermo Fisher Scientific), followed by transfer to nitrocellulose membrane (GE Healthcare Life Sciences) using an X10 Blot Module (Thermo Fisher Scientific) according to the manufacturer’s instructions. Membranes were stained with Ponceau solution to confirm transfer and even loading.
Membranes were blocked in 4% milk in 1x PBS 0.1% Tween (1xPBS-T) with shaking overnight at 4°C, before incubation with the appropriate antibodies (Santa Cruz sc-40 and sc-9996).
Signal was visualized by incubation with Amersham ECL Prime, on Amersham ECL Hyperfilm (both GE Healthcare Life Sciences), developed with a Compact X4 Automatic Processor (Xograph Healthcare Ltd).