Step-by-Step Visualized Ultra-Fast Western Blot

Experimental Procedure

Objective:

1. To experience the Western Blot experimental procedure and understand the value of step-by-step visualization in protein immunoblotting technology.
2. To familiarize with the functions of the imaging analyzer in result recording, image optimization, band quantification, and quantitative normalization.

Experimental Samples:

1. • Marker Standard 1: Precision Plus Protein™ All Blue Standards --- Tube labeled "B"
   • Marker Standard 2: Precision Plus Protein™ Unstained Standards --- Tube labeled "U"
   • Hela cell lysate: Original --- Tube labeled "F1"
   • Hela cell lysate: 1:2 dilution --- Tube labeled "F2"
   • Hela cell lysate: 1:4 dilution --- Tube labeled "F3"
   • Hela cell lysate: 1:8 dilution --- Tube labeled "F4"
   • Hela cell lysate: 1:8 dilution --- Tube labeled "F5"

2. Preparation of test samples: Starting from the provided F1 (already mixed with Loading Buffer), perform serial dilutions as shown in the table below:

   Tube Label	Content
   F2	15uL Loading Buffer + 15uL F1
   F3	15uL Loading Buffer + 15uL F2
   F4	15uL Loading Buffer + 15uL F3
   F5	15uL Loading Buffer + 15uL F4

   (Note: UTF is untreated fish meat extract.)

3. Place F1, F2, F3, F4, and F5 in a water bath for high-temperature denaturation at 95°C for 5 minutes. Afterward, remove the samples and arrange them in the loading order.

Gel Electrophoresis Steps

Electrophoresis - Precast Gel Group

1. Take out a precast gel (4-20% Stain-Free Mini-PROTEAN® TGX™ 10-well gel), remove the sealing tape at the bottom, assemble it into the Mini Tetra electrophoresis module, and then remove the comb.
2. If running only one gel, use a buffer dam or a discarded TGX gel with sealing tape to seal the opposite side of the module.
3. Fill the inner chamber of the assembled electrophoresis module with electrophoresis buffer (1x TGS Buffer) and check for leaks.
4. Remove the comb, gently rinse the sample wells with a plastic pipette (or 1 mL tip) to remove bubbles and straighten the wells. Load the samples and standards in the order shown below:
   

   Well	Volume	Sample Name
   1	10 ul	All Blue Standards ----B
   2	10 ul	Unstained Standards ---U
   3	10 ul	Hela-1 （F1）
   4	10 ul	Hela -2 (F2)
   5	10 ul	Hela -3 (F3)
   6	10 ul	Hela -4 (F4)
   7	10 ul	Hela -5 (F5)
   8	10 ul	Hela -5 (F5)
   9	10 ul	Unstained Standards ---U
   10	10 ul	All Blue Standards ----B

5. Place the loaded electrophoresis module into the buffer tank and fill the tank with the same buffer to the appropriate level.
6. Set the electrophoresis conditions to 250-300 V constant voltage for 15-20 minutes. If possible, place the electrophoresis tank on a stirrer with a stir bar for temperature equilibration.
7. After electrophoresis, remove the gel from the cassette, place it in water, and cut a corner for marking. Use the W5000 imaging system to capture the blotting results.
8. Prepare for the transfer step.

Protein Blotting Experimental Steps --- Transfer Section

Using Transfer Instrument for Rapid Transfer:

Mini Transfer Packs Midi Transfer Packs

The anode stack is placed on the right end (Bottom+). Use the right finger hole for removal.

1. Place the membrane and lower consumables (anode) on the transfer slot.
   • Use a specialized roller to remove bubbles.
   • For 1 small or medium gel, place it in the center of the transfer slot.
   • For 2 small gels, place them side by side without overlapping.
2. Place the electrophoresed gel on top of the membrane.
   • No gel equilibration is required before transfer.
   • Use a specialized roller to remove bubbles.
3. Place the upper consumables (cathode) on top of the gel.
4. Use a specialized roller to remove bubbles.
5. Close the transfer lid and lock it by rotating. Insert the correctly assembled transfer cassette into the Trans-Blot Turbo system to begin the experiment.
6. After powering on, select transfer instrument from the home screen's lower right corner to access preset programs. Choose 2 MINI OR 1 MIDI GEL, which sets the transfer conditions to [Mixed MW, 25V, 2.5A, 7 minutes]. Press "RUN" to start.

W5000 Multi-Functional Imaging System Demo Standard Operating Procedure

--- Immunodetection Section

1. After the transfer process, remove the PVDF membrane and check the transfer efficiency by verifying if the pre-stained protein standards have transferred onto the membrane.
2. Cut a corner of the membrane for marking.
3. Place the transferred PVDF membrane (gel-contact side up) into blocking buffer and incubate on a shaker for 1 hour.
4. Replace with 50 mL of primary antibody blocking solution and incubate for 1 hour at room temperature.
5. Rinse with wash buffer 3 times, 5 minutes each, on a shaker.
6. Replace with 50 mL of secondary antibody blocking solution + Strep-Tactin HRP and incubate on a shaker for 15 minutes.
7. Repeat the rinsing steps 3 times, 5 minutes each, on a shaker.
8. Perform chemiluminescent detection using freshly prepared Clarity ECL substrate solution.

   Reaction mixture =0.5 mL A (white bottle) + 0.5 mL B (black bottle).

9. Mix well and apply evenly to the membrane surface (use a pipette tip for even spreading). Allow the reaction to proceed for 1--3 minutes, then remove excess liquid without letting the membrane dry.
10. Capture the blotting results using the W5000 imaging system: Use the Chemi application in the Analysis Software with multiple cumulative exposure times (10 seconds, 4 images).
11. Save the results file and analyze the data using the Analysis Software.