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Student Resources: Student Projects: Abram Botvinnik
Description of the CSEM Research Project   view PDF

Introduction

This project is a research of Control system for Western blotting processor (WBP). The Western blotting process is widely used in biochemical and medical labs to detect presence of specific proteins. Moreover, these proteins can be seen by the color that they produce when proper reagents, in particular antibodies react with. This process makes it possible to find proteins in complex mixture, which would otherwise be a proverbial needle in haystack. Therefore, the Western blotting process is used in Medicine for diagnosis because of correlation between specific proteins in human blood or urine and some diseases, for instance different kind of cancers. It is used in Zoology, Botany, and the Food Science to demonstrate the presence and quantity of target proteins in the animals and plans that are studied. This process is used in Immunology and Microbiology to show which antibodies binds to which proteins in the studied organisms. In Biochemistry and Molecular Biology, it is used to help develop and discover new proteins. This technique is used advantageously in the cloning of genes [1].

An essential and most effort-consuming step in this process is treatment of membranes with numerous solutions over a prolonged period of time. Currently, it is done manually and this results in an inefficient use of time of the lab staff and makes it impractical to perform these tests overnight. Therefore, biochemical and medical labs need an inexpensive automated device with advanced features (more simultaneous tests with the larger number of used antibodies).

The Purpose of this Project

We participated in prototyping an advanced version of such device in cooperation with the group of the biochemical lab of Boston University (BU). Our part was designing the Control system. We are developed the Control system on the base of the Programmable Logical Controller (PLC). The PLC is a microcomputer used for automation of real-time processes. Using PLC for prototyping a new device is very convenient due it technical capabilities, flexibility, and low cost.

Method

The device is supposed to be used in biochemical and medical labs for Western blotting processing gel matrix in trays. This process has several steps. A prepared sample of solution with proteins by electrophoresis transfers proteins to gel matrix membrane. Blocking keeps these proteins on the membrane. Washing washed out excess parts of reagents. Antibodies react with desired proteins and activate color reaction. The list of operations and their sequence is shown in Table 1.

The device (12 ² ´ 12 ² ´ 6 ² ) consists of:

a hydraulic gravity feeding system providing storage of the reagents (non-aggressive water-based lukewarm fluids), their dosing and delivery to the trays;

a hydraulic gravity discharging system providing transportation of wasted blotting reagents from the trays to the sewer system and primary antibody collecting containers for re-using. (See Fig.1 Functional diagram);

a hydraulic cleaning system providing washing all containers and trays after processing;

a mechanical system providing reciprocate movement of the trays;

a control system providing data input and the turn on and off commands for solenoids, electric motor, and cleaning pump in the requested logical and time succession (see Fig.2 Electrical Diagram).

The Control system for WBP is based on the programmable logical controller (Fig.2). In order to choose appropriate type and model of PLC for our device, we got information from and contacted with almost of well-known PLC manufactures, such as Allen – Bradley, Siemens, Toshiba, Mitsubishi, Texas Instruments, and etc. We took in consideration many parameters of the PLC: price, sizes, (PLC should be installed inside the device), the number of outputs and their type (relays, transistors, etc.), output voltage and current, type of the power supply (AC or DC), user’s interface (display, indicator lamps, functional switches), method of programming (special programmer or supporting software for PC), etc. Eventually, we chose the world smallest Visual KV series PLC of the Keyence Corporation (Japan) [2].

The Western blotting process has three work stages: Data Input, Process, and Cleaning. The Process consists of several operations. We developed and programmed the Data input for each WBP operations (Table 1). As an operator display we use the Operator Interface Panel KV – K20 that has 4 lines ´ 20 digits display, four indicator lamps, and four switches [3]. To input the data into the PLC for each WBP operation we design several operator screen windows (Table 9). All entered data are automatically checked and in case of error the operator receives an error message on the display (see Table 9).

To program the PLC we used a special programming method (language) an Extended Ladder Diagram [4] on a personal computer with installed the Keyence Support Software [5] for the Visual KV Series PLC. Then program were downloaded to the PLC where it was stored.

Conclusion

For this project, we did following:

  1. Developed the WBP structure (the Functional diagram), work stages, operations, their sequence, algorithms, and timing diagrams.
  2. Developed the Control system for WBP, Electrical diagram, device outputs and their links to the executive elements: valve solenoids, electrical motor, and cleaning pump.
  3. Programmed PLC. Developed and programmed the Data input stage; programmed each of the Process stage operations and their sequence, and the Cleaning stage operations as well.
  4. Developed the Operator Manual: how to input data and how to control device work using switches, indicator lamps, and operator screens on the Operator Interface Panel KV – D20.
  5. Developed and provided the testing phase. All programs were tested and downloaded into PLC memory, where it will be stored. Then the PLC was connected to the WBP and tested, at first without fluids, then with fluids. “The performance of the device is excellent.” (See BU School of Medicine letter).

Recommendation for the Future

The next step of this project is advertising the device and collecting possible recommendations. Then should be done necessary device design and program corrections.

List of Figures and Tables

Fig. 1. Functional Diagram

Fig. 2. Control System Electrical Diagram

Table 1. Data Input for each Process Operation

Table 2. Process Operation Parameters

Table 3. Device Outputs

Table 4. Blocking Operation Timing Diagram

Table 5. Typical Cycle Timing Diagram for Washing Operations

Table 6. Primary Antibody (PA) Operation Timing Diagram

Table 7. Secondary Antibody (SA) Operation Timing Diagram

Table 8. Cleaning Timing Diagram

Table 9. Operator screen windows

Reference:

  1. Hainsworth, James, Western Blotting Report, 2000.
  2. Visual PLC Catalog , Keyence Corporation, Japan, 1999.
  3. User’s Manual, Operator Interface Panel KV–D20 , Keyence Corporation, Japan, 1999.
  4. The Visual KV Series User’s Manual, Programming, Keyence Corporation, Japan, 1999.
  5. The Visual KV Series User’s Manual, Support Software, Keyence Corporation, Japan, 1999.