6. Immobilization of antibody onto gold surface
6-1. Using batch system
Procedures:
1.At first, carefully clean the gold surface with dry air.
2.Set flow channel sheet (transparent sheet) onto the gold surface.
3.Add appropriate volume of antibody (5.0 µl) onto flow channel.
4.Incubate antibody containing chip onto rocking plate for 30-60 min at RT.
5.Carefully rinse with distilled water and remove water using cloth towel (Bemcot).
6.Again rinse with PBS and remove solution with Bemcot (or any soft towel / tissue that can absorb liquid).
7.Add appropriate volume of blocking buffer (0.5% casein or 1 mg/ml of BSA in PBS), and incubate onto rocking plate for 30-60 min at RT.
8.Rinse with distilled water and set PDMS for continuous flow.
 Click image to enlarge |
Note: After antibody immobilization onto gold surface, a completely dried gold surface may affect on antibody activity. Therefore, it is much better to apply blocking solution as soon as possible just after rinsing with water and PBS.
- The incubation time for antibody immobilization and blocking solution differs from antibody to antibody.
Antibody immobilization techniques are varied with antibody's size, shape, physical and chemical properties, and the diversity of applications for protein arrays. Therefore, users need to standardize the condition of antibody immobilization onto gold surface.
About antibody:
Polyclonal antibody contains several active sites for antigen binding region. There is a good possibility to see the antigen –antibody interaction. However, sometimes the non-specific binding response may be higher for polyclonal antibody because they may have sequence homology with other proteins. Considering those points, monoclonal antibody gives a minor non-specific response since it contains only one antigen-binding site that is very specific for particular substrate.
Blocking solution:
There are several solutions that are used for blocking the non-specific response of protein detection. The most common blocking solution is BSA (Bovine Serum Albumin) in PBS. However, the protein that has similar sequences with BSA may partially block the antigen binding regions with BSA solution (blocking solution). At this point, there are other blocking solutions that are also used for blocking in protein identification.
#Blocking solutions are-
1.BSA solution-commonly used for the detection of all proteins except that have similar sequences with BSA.
2.Casein solution- used for all proteins.
3.Protein free blocking buffer- it is commercially available blocking buffer for protein detection.
4.Skim milk-also used as a blocking reagent.
Precaution:
- Carefully apply antibody without any scratching or loss of gold particle by pipetting.
- A vigorous washing with micropipette sometimes detaches antibody from gold surface, therefore, carefully and slowly rinse the surface with water or buffer.
- Before setting PDMS onto gold chip, be sure that neither water nor solution remains onto the chip.
- Sometimes bubbles appear during applying flow system. To avoid this problem, at first, completely remove air from flow tube (FEP tubes), then fill with buffer and finally put into PDMS hole.
Catalog No. Description 012072 Handy SPR PS-0109 Components Transmission cable Power cable Software 011870 Matching oil 011869 Gold chip 011871 PDMS flow cell
