User Manual

Section 4

Protein Blotting

4.1 Immunoassay Procedure

Detailed instructions, including a comprehensive troubleshooting guide, for performing

immunoassays are included in the Immun-Blot

instruction manuals.

1. Assemble the Bio-Dot apparatus as described in Section 3. Prewet the membrane prior to

placing it in the apparatus. Nitrocellulose membranes are prewetted in TBS; nylon

membranes, such as the Zeta-Probe membrane, are prewetted in distilled water (see

Section 9 for solution preparation). Make sure that all the screws have been tightened

under vacuum to ensure that there will not be any cross-well contamination.

Notes: Zeta-Probe membranes must be removed from the Bio-Dot apparatus after the

antigen is immobilized. The blocking and other incubation steps should be carried out in a

separate container. Zeta-Probe membranes require more stringent blocking conditions,

using 5% (w/v) BLOTTO or 3% (w/v) gelatin in 1x TBS, which cannot be filtered through

the membrane using the Bio-Dot apparatus.

2. Rehydrate the membrane to ensure uniform binding of the antigen. Use 100 µl TBS per

well for nitrocellulose membranes. Use 100 µl distilled water per well for Zeta-Probe

membranes.

3. Adjust the flow valve so that the vacuum chamber is open to air (flow valve setting 2,

Figure 3). Fill the appropriate wells with antigen (protein) solution using any volume up to

500 µl per well. Multiple applications of antigen to a sample well are possible, but the

most rapid and efficient use of the apparatus is achieved by applying the required amount

of antigen in a minimal sample volume.

4. Allow the entire sample to filter through the membrane by gravity flow. Make sure that the

flow valve is positioned at a level below the sample wells to ensure proper drainage during

filtration applications. This passive filtration is necessary for quantitative antigen binding.

Each well should be filled with the same volume of sample solution to ensure homogenous

filtration of all sample wells. Generally, it takes 30–40 minutes for 100 µl of the antigen

solution to filter through the membrane. If antigen is very dilute, and it is necessary to

ensure that all proteins in the applied sample are filtered through the membrane, an

optional wash step can be performed. To perform this wash, add an aliquot of TBS equal

to the original sample volume to each sample well. Allow this material to passively filter

through the membrane by gravity filtration. (If the membrane is going to be removed from

the apparatus following binding of antigen, proceed to step 6 and follow the instructions

for the wash step. The wash step should be performed prior to disassembling the apparatus

to ensure that all antigen is removed from the drain ports underneath the membrane.)

5. After the antigen samples have completely drained from the apparatus, add 200–300 µl

of the blocking solution to each well. Allow gravity filtration to occur until the blocking

solution has completely drained from every well. This step should take approximately 60

minutes. Do not apply vacuum to speed up this step, as it will lead to poor assay results.