TITLE: Method for detecting acetylated PD-L1 in cell lysates
AUTHORS: Frances Middleton-Davis, Ashley Davis, Kim Middleton

[DESCRIPTION]
Here we present a method that allows detection of acetylated PD-L1 and is applicable to a wide range of cell lines. The method captures >90% of acetylated PD-L1 species, is semi-quantitative and simple to perform in any lab equipped with tissue culture and western blot equipment. The method involves processing cells in a lysis buffer that has been optimized for efficient immunoprecipitation (IP) of acetylated species, an IP enrichment step utilizing an acetyl-lysine affinity matrix and western blot detection of both total and acetylated PD-L1 on the same blot. This technique compliments the alternative IP approach utilizing a PD-L1 antibody as the IP reagent and an anti-acetyl lysine antibody as the detection reagent. However, because the protocol described here enables the detection of both total and acetylated PD-L1 on the same blot, this method has the advantage of allowing quantitation of the percent of PD-L1 that is acetylated, an important parameter for mechanistic interpretation. 

The method described here utilizes beads that are covalently linked to the affinity antibody, resulting in extremely clean IP results. Western blots can be re-probed with a pan anti-acetyl lysine antibody to visualize the total protein acetylation profile in any given lysate, a property that is useful when examining PD-L1 acetylation in the presence of HDAC inhibitors or other treatments affecting global acetylation.


[BEFORE_START]
The IP assay takes 48h.  Extensive tests were performed to confirm that the lysis buffer in this protocol maintains the stability of PD-L1 over at least a 72h time course, however, it is recommended to determine lysate stability with your specific cell lysate prior to carrying out the IP assay.

Each IP requires 0.5-1.0 mg of lysate protein. If the lysate protein yield from your tissue culture is not known, we recommend processing a "test plate" following the CELL LYSATE PREPARATION section below.  We recommend using 150cm2 plates for cell growth. As a rough guide, 50% confluent cell culture will generally yield 0.3 mg (e.g. Swiss 3T3)  to 2 mg (e.g. HeLa) from this size growth vessel.

[STEPS]
SECTION: IMMUNOPRECIPITATION ASSAY
None. Resuspend lyophilized Acetyl-Lysine affinity and Control beads in 500µl of 50% glycerol in MiliQ water and rotate for at least 1h at 4°C to rehydrate.60 min     

SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2. ACETYL-LYSINE IP: BUFFERS AND EQUIPMENT


Bead Wash Buffer: 100 ml, store at room temp.
 

Bead Elution Buffer: 10 ml, store at room temp.
 

Reagents and Equipment
Acetyl-Lysine Affinity beads
Mouse IgG Control beads
beta mercaptoethanol
Microfuge at 4°C
Rotator at 4°C equipped to hold microfuge tubes
Multipurpose mini spin columns (optional)

SECTION: IMMUNOPRECIPITATION ASSAY
1. Place supplemented Bead Wash Buffer on ice.
SECTION: IMMUNOPRECIPITATION ASSAY
2. Each assay requires 50µl of bead suspension. Aliquot the required number of beads into the required number of tubes.
SECTION: IMMUNOPRECIPITATION ASSAY
4. When the beads are ready, thaw lysates in a room temperature water bath.
SECTION: IMMUNOPRECIPITATION ASSAY
5. Centrifuge lysates as soon as they are all thawed. Centrifuge at 4°C in a microfuge at 14,000xg for 5m to ensure any degraded protein is pelleted separately from the sample.
SECTION: IMMUNOPRECIPITATION ASSAY
6. Save a small amount of lysate (~20µl) to run as a western input lysate control. Add 5µl of 5x SDS buffer and save for western analysis at -20°C.
SECTION: IMMUNOPRECIPITATION ASSAY
7. Carefully (so as not to disturb any centrifuged pellet) pipette 1mg of lysate and add to each tube containing beads (both control and acetyl).
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8. Incubate the tubes on a rotating platform at 4°C for 24h.1440 min 

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9. Collect beads by centrifugation at 5000xg for 1m at 4°C.
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10. Aspirate off as much supernatant as possible without disturbing the beads.
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11. Wash beads in 1ml of bead wash buffer (inhibitors are not necessary at this stage) for 10m each on a 4°C rotating platform at mid to high speed.
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12. Complete the wash step four more times (five washes total).
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13. After the final wash, completely remove the buffer supernatant with a pipette. Minimal disruption of the bead pellet is necessary.
 

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14. Add 28µl of bead elution buffer and resuspend the beads by gently tapping and flicking the side of the tube.
 


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15. Incubate beads in elution buffer at room temperature for exactly 5m.
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16. Gently pipette each bead suspension to one of the spin columns.
 

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17. Place the spin column in a fresh collection tube and centrifuge at 10,000xg for 1m at room temperature to collect the IP sample.
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18. Add 2μl of mercaptoethanol to each sample and mix well with a pipette.

SECTION: IMMUNOPRECIPITATION ASSAY
19. Samples can then be stored at -20°C.
SECTION: GEL ELECTROPHORESIS
21. Run gel(s) at 120V for 2h.120 min 

SECTION: WESTERN BLOT PROBING
29. DO NOT DRY BLOT after transfer. Move blot out of apparatus and go directly to block.
SECTION: WESTERN BLOT DEVELOPMENT
40. Develop blots with Super Signal West Dura Ultra HRP reagent. Use 4mL of 1X reagent per blot. Incubate for 5m at room temperature with constant shaking.
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22. While the gel is running, make 2L of cold western transfer buffer according to the recipe below.
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23. Get Western blot transfer tank(s) and place them in a cold room to cool down (4°C).
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22.1. Place the buffer in the -20°C freezer to cool down.
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24. When the gel is completed, soak in ~100mL of cold western transfer buffer for 15m with gentle shaking.  This can be done at room temperature. 
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25. Cut blot-sized piece(s) of PVDF membrane and soak these for 1m in methanol.
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26. Rinse PVDF membrane(s) briefly in water and transfer them into western transfer buffer until ready to use.
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27. Assemble gels into the western blot apparatus.
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28. Blot gel(s) at 20V for 17 hours (overnight) in a cold room (4°C).
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30. Block the blot in 5% milk in TBST. Incubate for 1 hour at room temperature with constant shaking.60 min 

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31. Rinse briefly in TBST.
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32. Locate the PDL1 antibody and dilute it to a 1:1500 concentration in TBST with 3% milk.
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34. Incubate the primary antibody for 1.5 hours at room temperature with constant shaking.  
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35. Before removing from the primary antibody, make a 1:25,000 anti-rabbit secondary antibody solution in TBST with 3% milk.
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36. Wash blot(s) 2 times in TBST for 3 minutes
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37. Add 10 ml of diluted secondary antibody.
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33. Add 10ml of diluted primary antibody.
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38. Incubate the secondary antibody for 40 minutes at room temperature with constant shaking.  
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39. Wash the blot 6 times for 10 minutes each in 50mL of TBST.
SECTION: WESTERN BLOT DEVELOPMENT
41. Develop western on the following settings for 1m, 5m, and 10m:
- High/Medium Speed/Resolution
- Show Saturation
- No Autoconstrast
SECTION: WESTERN BLOT DEVELOPMENT
42. Print all pictures and record the date and exposure time on each of them.
SECTION: GEL ELECTROPHORESIS
20. Prepare gel box(es) with 1x SDS Gel Running Buffer and 4-20% Tris-glycine Gel(s).
 

SECTION: IMMUNOPRECIPITATION ASSAY
3. Wash beads in Bead Wash Buffer 
None. 
SECTION: CELL LYSATE PREPARATION
1. 
LYSATE PREPARATION: BUFFERS AND EQUIPMENT

Determine the volume of lysis and dilution buffer needed to process your cells. 

For each 1 mg of lysate protein you will need:
                                                                                    300 μl    supplemented Lysis Buffer
                                                                                    1.2 ml    supplemented Dilution Buffer

Make buffers as detailed below just prior to processing cell lysates. Buffer volumes should be scaled according to needs, the recipes are for 1 ml of each buffer. Each 1ml of Lysis buffer requires 4 ml of Dilution buffer.

Supplemented Lysis Buffer (1 ml): Store at room temperature, use within 3h
 

Supplemented Dilution Buffer (1 ml): Store on ice, use within 3h
 

1:4 Dilution Buffer (1 ml): Store on ice, use within 3h
 

Reagents and Equipment
Phosphate Buffered Saline (PBS) pH 7.0
Microfuge tubes
Cell lifter
BlastR filters (each filter can process up to 1 ml of lysed cells)
Liquid nitrogen (to snap freeze lysates)

SECTION: CELL LYSATE PREPARATION
1.1. CELL LYSATE PREPARATION: PROCESSING CELL LYSATES

The required number of cells should be grown and treated as required (see Before Starting section above).
 

SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.1. ACETYL-LYSINE IP: IMMUNOPRECIPITATION STEP

 

Aliquot 50 μl of Acetyl-lysine affinity bead slurry (approx. 50 μg conjugated antibody) for the required number of IP reactions.
 

SECTION: SDS-PAGE
3. 

1.    Boil samples for 5 minutes before running on SDS-PAGE.
 

SECTION: WESTERN BLOT PROCESSING AND ANALYSIS
4. WESTERN BLOT: BUFFERS AND EQUIPMENT


Western Transfer Buffer (1L per gel), store at room temp.
 


Tris Buffered Saline/Tween (TBST Buffer) (10L). store at room temp.
 


 Antibody Block Buffer: 50ml, store at room temp.
 

 
 
Antibody Dilution Buffer: 50ml, store at room temp.
 

SECTION: WESTERN BLOT PROCESSING AND ANALYSIS
4.1. WESTERN BLOT: PROCESS & ANALYSIS

Transfer the proteins to PVDF membrane using the Western Blot Transfer Buffer. Transfer at 20V for 17h.

1020 min 
 

SECTION: CELL LYSATE PREPARATION
1.2. Remove culture media and wash cells twice with 10 ml each of room temperature PBS buffer.

 

SECTION: CELL LYSATE PREPARATION
1.3. Add the appropriate volume of Supplemented Lysis Buffer determined in STEP 1 and harvest cells using a cell lifter.  The lysate will become highly viscous, due to nuclear lysis. If processing multiple plates for a given condition, the lysates can be pooled at this point (use a snipped 1 ml pipette tip).
SECTION: CELL LYSATE PREPARATION
1.4. Use a snipped 1 ml pipette to transfer up to 1 ml of crude lysate per BlastR filter and harvest the flow through into a 15 ml tube on ice. The filter step is necessary to reduce sample viscosity (by removing DNA from the lysate).
SECTION: CELL LYSATE PREPARATION
1.5. Record the volume of the lysate and add 4 volumes of Supplemented Dilution Buffer. Mix gently.
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1.6. Immediately take the protein concentration.


 

SECTION: CELL LYSATE PREPARATION
1.7. Dilute the lysate to 1 mg/ml using 1:4 Dilution Buffer and immediately dispense the lysate into 1 mg aliquots.
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1.8. Snap freeze the lysate aliquots in liquid nitrogen and store at -80°C.
 

SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.2. Aliquot 50 μl of mouse IgG control bead slurry (approx. 50 μg of conjugated antibody) for the required number of IP control reactions. 
SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.3.  Wash the beads once with 1 ml each of PBS. Collect beads by centrifugation at 2000 x g for 1 minute at 4°C and remove PBS.
SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.4. Add 1 mg of appropriate lysate to corresponding beads and incubate with rotation (approx. 25-30 rpm) at 4°C for 24h.

1440 min 

SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.5. Collect beads by centrifugation at 2000 x g, 1 minute, 4°C . Save the supernatants in clearly labeled tubes on ice

 

SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.6. Wash the beads by adding 1 ml of Bead Wash Buffer and rotating (25-30 rpm) for 10 minutes at 4°C.

10 min 

SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.7. Collect beads as above and repeat the bead wash step 4 more times.

10 min
10 min 
10 min 
10 min 

SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.8. After the final wash, make sure that the wash buffer is completely removed from the beads. A fine bore pipette tip can be used for this step.  Failure to remove all wash buffer will reduce effectiveness of the elution buffer and will result in volumes too large for gel analysis.
SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.9. Elute the acetylated proteins by adding 27 μl of Bead Elution Buffer and incubating at room temp. for exactly 5 minutes. The tubes can be gently flicked to mix the beads and elution buffer. Do not use a pipette for mixing.
SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.10. Collect beads by centrifugation at 5000 x g for 1 minute and remove supernatant containing acetylated proteins.

 

SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.11. Add 2 μl of beta mercaptoethanol to each tube and place at -80°C.  These samples are the 24h IP elutions.
SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.12. Take the supernatants saved in step 2.5 and add a fresh 50 μl aliquot of appropriate beads to each.
SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.13. Incubate for a further 24h with rotation at 4°C.  These will be the 48h IP samples.

1440 min 

SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.14. Collect the 48h IP beads as above.  Lysate supernatants can be discarded.
SECTION: ACETYL-LYSINE IMMUNOPRECIPITATION (IP)
2.15. The 48h IP elutions should be pooled with the corresponding 24h elutions. Samples can now be processed for western blot analysis.
SECTION: WESTERN BLOT PROCESSING AND ANALYSIS
4.2. Block the membrane for 2h in Antibody Block Buffer.

120 min 

SECTION: WESTERN BLOT PROCESSING AND ANALYSIS
4.3. Dilute primary antibody 1:1,500 in Antibody Dilution Buffer and incubate with shaking for 1.5h at room temperature.  We use 10 ml of diluted primary antibody for a mini-gel sized blot.

90 min 

 

SECTION: WESTERN BLOT PROCESSING AND ANALYSIS
4.4. Wash blot 2 x for 5 minutes each in TBST.

5 min 
5 min 

SECTION: WESTERN BLOT PROCESSING AND ANALYSIS
4.5. Dilute secondary-HRP antibody 1: 25,000 in Antibody Dilution Buffer and incubate with shaking for 40 minutes at room temperature.  We use 10 ml of diluted primary antibody for a mini-gel sized blot.

40 min 

SECTION: WESTERN BLOT PROCESSING AND ANALYSIS
4.6. Wash the blot in 50-100 ml of TBST for 10 minutes.

10 min 

SECTION: WESTERN BLOT PROCESSING AND ANALYSIS
4.7. Repeat wash 5 more times.

10 min 
10 min 
10 min 
10 min 
10 min 

SECTION: WESTERN BLOT PROCESSING AND ANALYSIS
4.8. Develop the blot using Thermo Fisher Super Signal West Dura Chemiluminescent reagent, 10 minute incubation at room temp. in 4ml of reagent per mini gel blot is recommended.

SECTION: WESTERN BLOT PROCESSING AND ANALYSIS
4.9. Develop the blot and save the image as a tiff file. Analyze the PD-L1 signal (approximately 50 kD) using ImageJ software.