BiP | Lumenal-binding protein (goat antibody)

Application example

5 µg of total protein from A.thaliana (1), H. vulgare (2),  Z.mays (3),  S. oleracea (4), extracted with Agrisera PEB extraction buffer (AS08 300) were separated on  4-12% SDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in  for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-goat IgG horse radish peroxidase conjugated, from  Agrisera AS09 605) diluted to 1:50 000  for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL  detection reagent according to the manufacturers instructions.  Exposure time was 5 seconds.



Immunofluorescent localization of BiP in suspension cultures of Arabidopsis thaliana (Landsberg erecta cv. MM1) using goat anti-BiP polyclonal antibodies (AS09 615) and donkey anti-goat IgG DyLight®488 conjugated secondary antibodies (AS10 1116, Agrisera). 

Material: suspension cultures of Arabidopsis thaliana ecotype Landsberg erecta cv. MM1)
Fixation: Packed cell volume to fixer ratio: 250 µl : 5ml
Fixer composition and buffer: 4% (w/v) paraformaldehyde (freshly prepared as 8% stock and 0.2 µm filtered) 0.01% (v/v) Triton-X100 in Phosphate Buffered Saline (PBS), pH 7.4 (2x stock, 0.2 µm filtered)
Container and method: in 6 cm Petri dish, gentle shaking at room temperature (RT)
Duration: 25 min.
Hydrophilization: No
Cell wall digestion: Yes
Packed cell volume to enzyme ratio: 100 ul : 2ml Enzyme composition: 1% Cellulase (chromatically purified, powder, Worthington) 1% Pectinase (protease free, liquid, Sigma) Buffer: 0.5% (w/v) MES buffer, pH 5.6 Container and method: in 2 ml microfuge tube by rolling at room temperature (RT) Duration: 30 min.
Membrane permeabilization: Triton-X100 (0.5%), 10 min/RT
Antigen retrieval: No
Blocking buffer: Fish gelatin (5% v/v)
Washing buffer: PBS
Primary antibody dilution and incubation time: 1:400, ON/4ºC
Secondary antibody: donkey anti-goat IgG DyLight®488 conjugated secondary antibodies (AS10 1116, Agrisera), 1: 600, 1h/RT
Co-staining of the nucleus (DAPI): Yes
Cell wall and nucleus staining: 100 ng/ml DAPI

Courtesy of Dr. Ferhan Ayaydin, Hungarian Centre of Excellence for Molecular Medicine (HCEMM), Szeged, Hungary

Protein or membrane sample should be treated at 70°C for 10 min before loading on the gel.

Antibody has a reduced reactivity to monocots in western blot.

BiP2 (Binding immunoglobulin protein) is localized in endoplasmic reticulum lumen (ER) and plays a role in protein assembly inside ER.  BiP protein is abundant under all growth conditions but its synthesis can increase under conditions that lead to the accumulation of unfolded polypeptides in endoplasmic reticulum (ER). Alternative name: AtBP2