Application Note 38

Western Blot Analysis
with Cell Samples Grown in Channel-µ-Slides
Polyacrylamide gel electrophoresis (PAGE) and subsequent analyses are commontools in biochemistry and molecular biology. This Application Note describes a
protocol for preparing cell lysates directly in the ibidi channel-µ-Slides and thesubsequent separation in an SDS-PAGE. The proteins are blotted to a nitrocellulose
membrane and the species of interest are analysed with immunochemistry. In ourexample, we show the detection of VE-cadherin in HUVECs.

Table of Contents:
1. Material ................................................................................................................ 1
2. Cell Cultivation ..................................................................................................... 2
3. Cell Lysis.............................................................................................................. 3
4. BCA Protein Assay............................................................................................... 4
5. SDS PAGE and Blotting ....................................................................................... 4
6. Antibody Labeling and Scan ................................................................................ 5

1. Material
Cell Culture
Minimum 1 x 106 cells
4 pieces

HUVECµ-Slide I0.4 Luer
Endothelial Cell Growth Medium
Supplement Mix (containing 2% FCS)PBS 1xAccutase
Optional: ibidi Pump System for flow conditioning of the cells

Cell Lysis
Cell Signaling (#9803)
1 mM PMSF in lysis puffer

Cell Lysis Buffer
PMSF (proteinase inhibitor)
PBS 1xFine dosage syringe with displacement
spike (1 ml) + 25-gauge needle
Reaction tubes 1.5 ml
Sonicator

BCA Protein Assay
Scientific)
96 well plate
Plate reader (562 nm)

BCA Protein Assay
Kit (Thermo_____________________________________________________________________________________________________
Application Note 38© ibidi GmbH, Version 1.0, 10.March 2015
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Application Note 38

Loading Buffer (Roti®-Load 1)
Heating Block with Shaker (90°C)
Gel (12% SDS)
Gel electrophoresis apparatus
power supply
Protein standard
Electrophoresis running bufferSDS-PAGE
K929.1; Carl Rothwith

Blotting
Blotting apparatus
Blotting buffer
Nitrocellulose membrane
Filter paper
Ponceau S solution
PBST20 ml
0.075% Tween20 in PBS
Antibody Labeling and Scan
For membrane incubation

Compact Rocker
Milk powder
PBST
Primary antibody
Secondary antibody
Immunofluorescence reader

0.075% Tween20 in PBS

2. Cell Cultivation
Before the lysis of the cells cultivate them in a ibidi µ-Slide according to yourexperimental setup. This Protocol was established with flow-cultivated HUVECs.
Therefore, µ-Slides I0.4 Luer are filled with a cell suspension of 2*106 cells/ml. The µSlides I0.4 Luer with seeded HUVECs are placed in an incubator for two hours toassure that most of the cells are adherent before the start of the flow cultivation. The
setup of a flow experiment to cultivate HUVECs under shear stress conditions is
described in Application Note 13. Four µ-Slides, which were flow-cultivated over aperiod of two days with a shear stress of 10 dyn/cm² (Figure 1), are used for
producing one cell lysate.

Figure 1: Flow-cultivated HUVECs at 10 dyn/cm² after a period of two days. The arrow indicates thedirection of the flow (Scale bar: 200 µm).
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Application Note 38

3. Cell Lysis
Work on ice to avoid degradation of proteins. Put PBS, cell lysis buffer, needle,syringe, and 1.5 ml reaction tubes on ice. Just prior to use, add 1 mM of PMSF to thelysis buffer. For each cell sample, use a minimum of four µ-Slides I0.4 Luer with aconfluent cell layer.
In order to minimize the volume of the resulting protein solution, a volume of 200 µl
lysis buffer is used for the first Slide to lyse the cells, and then reused for thesubsequent Slides.
1. Put the four µ-Slides on ice.
2. Wash every channel Slide three to four times with 120 µl PBS each, in order toremove the cell culture medium.
3. Start with one µ-Slide; keep the other slides filled with PBS on ice, while lysing
the cells of the first Slide.
4. Remove the PBS from the channel.
5. Draw up 200 µl of the lysis buffer with the syringe (Figure 2 A). Do not use theneedle here!
6. Plug the syringe into one Luer adapter of the empty channel Slide and fill up
the channel with the lysis buffer (Figure 2 B).
7. Incubate with the lysis buffer for five minutes.
8. Shear the cells out of the channel by moving the plunger quickly up and down.
Be careful not to squeeze out the cell lysate at the free Luer adapter of theslide. Check under the microscope to see if the cell layer is completely
washed out.
9. Fully remove the PBS from the next channel slide.
10. Draw up the entire cell lysate with the syringe and fill the next PBS-freechannel with the lysate. The residual lysate can be pipetted directly into aprecooled 1.5 ml reaction tube.
11. Repeat Steps 6 through 10 for all Slides.
12. After the treatment of the last Slide, fill the lysate into the previously used 1.5
ml reaction tube.
13. Slip the precooled needle onto the syringe and pass the lysate through theneedle 10 to 15 times. For a better cell disruption, you can also use asonicator.
14. Spin the extract in a microcentrifuge at 14,000x g for 10 minutes at 4°C.
15. Transfer the supernatant into a new, precooled 1.5 ml reaction tube. For future
processing, the lysate can be stored at -20°C.

Figure 2: A) Fine Dosage syringe with the displacement spike filled with 200 µl. B) µ-Slides I
filled by the syringe. Lyse cell layer in channel slide by moving the plunger up and down.

0.4Luer_____________________________________________________________________________________________________
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Application Note 38

4. BCA Protein Assay
The Pierce BCA Protein Assay Kit (Thermo Scientific) is used to determine the total
amount of protein. The total protein is quantified by a formulation that is based on
bicinchoninic acid (BCA) for a colorimetric detection. The assay should be performed
as described in the manufacturer’s instructions, and using a µ-Plate 96 well.

5. SDS PAGE and Blotting
A general protocol for lysate preparation, SDS PAGE, as well as membrane blotting,is described below. The optimal protocol for your protein of interest might differ fromthe one given below according to the characteristics of your protein.
1. Mix the desired protein amount with the sample buffer (at least 15 µg of total
protein is recommended).
2. Heat the sample at 90-100°C for 5 minutes and then cool down the sample
afterwards.
3. Load the complete sample onto an SDS PAGE gel. Add a (prestained) protein
ladder into an extra lane.
4. Set an appropriate constant voltage, depending on how many gels you run
and the gel’s thickness.
5. Stop the electrophoresis run when the dye front reaches the bottom of the gel.
The running time depends on the applied voltage.
6. Place the gel and nitrocellulose membrane separately in the blotting buffer for15 minutes.
7. Prepare the transfer stack (the membrane should be on the cathode and thegel on the anode side).
8. Transfer the proteins at a constant current. The duration and the current’s
height will depend on the blotting method.
9. Stain the membrane with Ponceau S to check the transfer quality.
10. Rinse the membrane three times for 10 minutes with PBST to remove thePonceau S staining.

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Application Note 38

6. Antibody Labeling and ScanTo detect the protein bands on the membrane a suitable labelling must be done. The
detection of the analyzed protein band was performed using fluorophore-conjugatedsecondary antibodies. Figure 3 shows an example of a fluorescence-labeled VEcadherin band.
During the mentioned incubation times of step 1 to 5 the membrane has to be shaked
gently.
1. Block the membrane for 1 hour at room temperature using a blocking solution(5% milk powder in PBST).
2. Incubate the membrane with the recommended dilution of the primary
antibody in a blocking solution overnight at 4°C.
3. Wash the membrane three times with PBST for a duration of 10 minutes eachtime.
4. Incubate the membrane with the recommended dilution of the fluorophoreconjugated secondary antibody in blocking solution for 1 hour at roomtemperature.
5. Wash the membrane three times with PBST for a duration of 10 minutes eachtime.
6. Dry the fluorescently labeled membrane between filter papers for at least 1hour.
7. Scan the dried membrane with an appropriate emission filter. The generated
digital image of the western blot allows for further data analysis.

Figure 3: Scan of the fluorophore-labeled VE-cadherin band at the expected size of ~140 kDa.

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