Blot Plate: Fast and easy 96-well dot blot assay

Advantages:

1. Save sample and antibody.  Only use 1-2 µl of primary or secondary antibody for each sample,  not 100s µl used in conventional methods.
2. Get results fast. 1 hour is enough for a regular dot blot assay using a labeled primary antibody.
3. Zero cross-contamination. Each well is a completely separated unit. There will be no leakage or diffusion between different samples. Signals are confined within each sample well. 
4. Stand alone device ready for use. No need to use vacuum pump, centrifuge, shaker or aspirator.

A Simple Dot Blot Protocol ( All you need to do is pipette ):

A comparison between a conventional dot blot method and Blot Plate: 

The Difference is Clear. 

No shaking. No vacuum. No aspiration. No more mess with a piece of delicate membrane. With Zoom Plate, all you need to do is sequentially add samples and reagents into the wells.  

In a typical Blot Plate assay, such as dot blot, direct ELISA, indirect ELISA, and other type of sandwich assays, 1-2 µl of a capture antibody or antigen is first spotted onto the membrane and let air dried for about 20-30 min. Afterwards, the membrane need to be blocked for 20-30 min. Additional reagents, such as antigen, primary antibody, and secondary antibody are added sequentially into the well and let react with the previously immobilized molecules for no more than 5 min. Unbound molecules will be rinsed away with rinsing buffer by the absorbing plug under the membrane. If the antibody is labeled with a fluorophore or a colored-agent, the plate can be examined immediately after the final rinse. If the antibody is labeled with an enzyme, such as HRP or AP, a small amount of enzyme substrate will be added onto the membrane to allow luminescent or chromogenic signal detection.

The result - zero cross contamination, low background, high signal/noise ratio, easy and accurate quantification, clean and presentable data images, all can be obtained within 1.5 hr, including reagent preparation time.  

Support Multiple Assay Formats:

Direct ELISA	Indirect ELISA	Direct Sandwich Assay	Competitive Assay

	Application Example 1: Chemiluminescent IL6 Dot Blot Assay

Blot Plate delivers fast result with superior sensitivity. 1 µl of IL6 with concentration at 0.1 - 100 µg/ml was spotted on a Blot Plate (cat# MGF16B) with 1.6 µm pore size glassfiber membrane. After 20 min blocking, the following reagents were added to the wells step by step: 2 µl of 2 µg/ml (1:500 dilution) primary antibody (5min), 50 µl rinsing buffer 1%BSA-PBST, 2 µl of  0.5 µg/ml   (1:2000 dilution) of HRP-conjugated secondary antibody (5 min), 200 µl rinsing buffer PBST,  and 10 µl of HRP ECL substrate. Chemiluminescent signals were measured in a gel imaging system. Error bars are standard deviations. R2=0.997. 

Conventional Dot Blot. As a comparison, we performed a conventional dot blot using a piece of nitrocellulose (NC) membrane, and the same IL6 standard, antibodies and ECL substrate as we used in the above Blot Plate experiment. The assay took more than 3 hours to finish. Due to significant cross-contamination and high background across the membrane, the 1 ng dots can barely be identified.  Assay detail: The NC membrane was blotted with 1 µl of IL6 with concentration at 0.1 - 100 mg/ml for 30 min, and blocked with 20 ml SuperBlock (Thermo Scientific) blocking buffer for 20 min. The membrane was then incubated with 20 ml 1:1000 dilution of mouse anti-IL6 antibody for 30 min,  and washed with PBST for 3 times, 5 minute each. After that, the membrane was incubated with 1:2000 dilution of anti-mouse IgG-HRP for 30 min, and washed with PBST fro 3 times, 5 minute each. 1 ml ECL substrate SuperSignal Dura was added onto the membrane and the image was taken in a Gel Imager.

	Application Example 2: Colorimetric Dot Blot Assay Using Au Nanoparticle

Colorimetric Immunoassay Using Au Nanoparticle. 1 µl Mouse IgG from 1-1000 µg/ml was blotted on three white Blot Plate strips with different filter membranes. After 20 min blotting, 50 µl 10% BSA was added for a 20 min blocking. 1 µl Goat anti-mouse IgG labeled with Au nanoparticle was added into each well and incubated for 5 min. Afterwards, the wells were rinsed with 50 µl PBST four times. Images were taken before and after the rinsing step. Distinctive red color can be clearly visualized against the white background. Intensity of the red color is correlated with the concentration of the dotted proteins. In this experiment, MCE membrane demonstrated a better detection sensitivity (10 ng) than the glassfiber membranes (100 ng) for colorimetric assay.

	Application Example 3: Chromogenic Dot Blot Assay

Chromogenic Immunoassay Using HRP-labeled Antibody. 1 µl Mouse IgG at 10 µg/ml and 1 µg/ml was blotted  on a Blot Plate strip (cat# MMC30W, MCE 3.0 µm pore size membrane). After 40 min blotting, 50 µl 10% BSA was added for a 20 min blocking, followed by a 20 µl 1% BSA-PBST rinsing. 2 µl 5 nM HRP-labeled goat anti-mouse IgG was added into each well and incubated for 5 min. Afterwards, the wells were rinsed with 50 µl PBST four times. 10 µl chromogenic HRP substrate (R&D Systems) was added onto the membranes for color development. Images were taken 10 min after substrate addition. Distinctive blue color can be clearly visualized against the white background. No substrate was added into the blank wells. 

Product Selection Guide:

 Note:  Detection methods -  FL: fluorescent;  CL: chemiluminescent; CM: colorimetric.

 Black or white:

Black wells are great for sensitive fluorescent and chemiluminescent detection, because black material absorbs stray lights and minimize the inter-well optical cross-talk. 

White wells are great for colorimetric signal reporting, including Au Nanoparticle, colored latex microspheres, chromogenenic HRP substrate, etc. Vivid color can be well presented against white background, easy for visual inspection and color imaging. 

White wells can also enhance chemiluminescent signal. If your CL detection system is not sensitive enough for black plates, you can try white plates. 

 Glassfiber, Nitrocellulose, MCE:

Nitrocellulose membranes are the most popular selection of matrix used in protein and nucleic acid blotting because of their high binding affinity, compatibility with a variety of detection methods (chemiluminescence, chromogenic, and fluorescence), and the ability to immobilize proteins, glycoproteins, or nucleic acids.

Borosilicate Glassfiber membrane offers fast flow rate, low fluorescent auto-fluorescence background, but with lower protein binding capacity. It is great for all general purpose assays with fluorescent, chemiluminescent, and colorimetric detections.  

Mixed Cellulose Ester (MCE) is a mixture of cellulose nitrate/acetate fibers. It has a smooth surface and is commonly used in diagnostic kit manufacturing.  It has higher protein binding capacity than glassfiber, generates stronger signal. It is great for chromogenic detection, in which color develops much faster (<5 min). 

Resources:

	Protocol: Cholorimetric Dot Blot Assay
	Protocol: Chemiluminescent Dot Blot Assay
	Protocol: Chemiluminescent Sandwich Assay
	Protocol: Fluorescent Direct Dot Blot Assay
	Protocol: 化学发光点印实验方法说明
	Application Note - Zoom Plate Dot Blot
	Blot Plate Specifications
	Comparison between Blot Plate membrane materials