Zoom Blot: Fast and Easy 96 Well Dot Blot

Fast and Easy Dot Blot Using Zoom Plate

Advantages:

  1. Save >99% 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.Optical 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 Zoom Blot: 

 

Conventional Methods

(per 8cm x 10cm membrane)

Zoom Blot

(per well)

Required equipment

membrane, vacuum pump, microfiltration apparatus, 

membrane holder box , shaker, clear wrap

Zoom Plate and pipetter only

Blotting

 1 µl antigen,  10 – 100 µg/ml

> 30 min, must be dry

1µl antigen, 0.1 – 100 µg/ml

20 min, don’t have to be dry

Blocking

20 ml blocking buffer

0.5 - 2hr

 50 µl blocking buffer

25 min

Primary antibody

20 ml or 200 µl/well  1:1000 dilution

0.5 - 2 hr

 2 µl 1:1000 dilution

5 min

Rinsing

20 ml rinsing buffer

3 x 5 min on shaker

 add 20 µl rinsing buffer

no waiting

Secondary antibody

20 ml or 200 µl/well 1:1000 dilution

0.5 - 2 hr

 2 µl 1:1000 dilution

5 min

Rinsing

20 ml rinsing buffer

3 x 5 min on shaker

 add 4 x 50 µl rinsing buffer

no waiting

ECL substrate

1-2 ml

10 µl

Detection

Wrap membrane in a clear film,

image in a membrane imager

 Directly put in an imager or a plate reader

 

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 Zoom Blot 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                                                                                                                  

Zoom Blot delivers fast result with superior sensitivity. 1 µl of IL6 with concentration at 0.1 - 100 µg/ml was spotted on a Zoom 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 Zoom 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 Zoom Blot Using Au Nanoparticle. 1 µl Mouse IgG from 1-1000 µg/ml was blotted on three white Zoom 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 Zoom Blot Using HRP-labeled Antibody. 1 µl Mouse IgG at 10 µg/ml and 1 µg/ml was blotted  on a Zoom 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.