TITLE: Blue native-PAGE of protein complexes in plant cells
AUTHORS: Hee-Kyung Ahn, Jonathan Jones

[DESCRIPTION]
Many protein complexes exist in cells, and these protein complexes are vital for cellular processes. However, much of our current analyses of protein-protein interaction mainly focus on binary interactions. Non-denaturing PAGE (polyacrylamide gel electrophoresis) provides an efficient method that enables analysis of multimeric protein complexes in vivo. In contrast to chromatography or sucrose fractionation methods, small amount of sample is required for analysis, and multiple combinations of samples can be tested simultaneously. Non-denaturing PAGE using the Coomassie G-250 dye, also known as blue native-PAGE resolves protein complexes according to their molecular weight and has been successfully used to identify protein complexes bound to membranes such as in mitochondria and chloroplasts. Recently, intracellular immune receptors in plants localizing in the cytoplasm, nucleus or plasma membrane were analyzed using the blue-native PAGE method. Here we share our method of resolving protein complexes from plant extracts or immunoprecipitated samples from plant cells on blue native-PAGE.

[BEFORE_START]
This protocol was developed over time by Hee-Kyung Ahn in the Jonathan Jones group. This method has been optimized mainly for detecting the oligomers of NRC NLRs in Solacaceae, but we have tested that this protocol also applies to NLRs in Arabidopsis seedlings and rosette leaves. For other proteins, additional optimization may be needed, and we are continuing to optimize the method for other NLR proteins. We hope this protocol will be useful, and help generate new discoveries in the plant biology community and beyond. If you have any suggestions or feedback regarding this protocol, please email hee-kyung.ahn@tsl.ac.uk.

[GUIDELINES]
* Although this protocol used the NativePAGETM system from Thermo Fisher Scientific, similar set-ups from different suppliers, as well as setting up your own gel without SDS works just as well.
* Ensure samples are always kept on ice, and tissue samples are frozen immediately, to prevent dissociation or aggregation of protein complexes.
* Protein extraction buffer can significantly affect protein complex stability as well as extraction. Salt concentration of buffer, type and concentration of detergent used, as well as concentration of DTT are all critical factors that determin protein exractabiligy. Therefore, optimal extraction buffer must be empirically tested for each protein of interest.
One way to check extractability of buffer is to test whether any proteins remain in the pellet after centrifugation of cell debris. Protein extracts using different buffer conditions can be loaded onto blue-native PAGE to compare which buffer condition is ideal for extracting protein complexes. 
However, not all buffer conditions ideal for extracting proteins will be ideal for preserving native states of protein complexes. The protein extraction buffer condition provided in this protocol is ideal for detecting oligomerization of NRC2, and related experimental data can be found in attachment.
* Concentration of plant extracts are critical for running blue native-PAGE. If bands appear smeary, it is likely due to high concentration of protein extracts leading to aggregation. Reducing the protein extraction concentration by dilution may resolve this issue. In particular, when using protein extracts, rubisco complex (~500kDa) should be clearly visible, in addition to bands at ~720kDa, and 1,000kDa for N. benthamiana extracts.
* Accumulation of protein complexes identified in BN-PAGE must correlate with protein accumulation observed on SDS-PAGE.
* 2 Dimensional SDS-PAGE should be performed to check that the protein bands observed on blue native-PAGE constitute of proteins that run on the same size on SDS-PAGE.
* The relevant position of the bands observed must be consistent throughout multiple replicates. This enables measuring the approximate size of protein complex with standard curve from the markers.

[STEPS]
SECTION: Sample preparation
1. The following cases describe sample harvest methods using trasiently-infiltrated Nicotiana benthamiana (4-5 weeks old), Arabidopsis seedlings (~2 weeks old), or Arabidopsis leaves (4-5 weeks old.)
SECTION: Protein extraction
2. Grind samples in 2 ml tubes using a tissue homogenizer (GenoGrinder), at 1200 rpm for 1 min . 
Samples should be kept frozen at all times, and put sample tubes back into liquid nitrogen immediately after sample grinding is complete.
 

SECTION: Separation of samples by Blue Native-PAGE
10. NativePAGE™ 3 to 12% pre-cast gel is used with XCellSureLock™ Mini-Cell system.

 

SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
22. After gels are run until dye fron reaches end of gel, gel cassette is pry open using cassette opening lever/gel knife.
Lift open all three sides of the cassette, and carefully cut both ends of the gel (side with wells and the opposite side where the dye front is).
SECTION: Protein extraction
3.  Open tube lids on ice, and add extraction buffer (see Materials, Buffer recipes).
The amount should be 200 µL , or 200 µL .

SECTION: Protein extraction
4. Vortex for 15 s .

SECTION: Protein extraction
5. Incubate sample on rotator (must be kept cold) for 10 min .

SECTION: Protein extraction
6. Centrifuge at 4°C, 13,000rpm, for 15 min .

SECTION: Protein extraction
7. Transfer supernatant to clean new tube, centrifuge again at 4°C, 13,000 rpm for 5 min .

 

SECTION: Protein extraction
8. Make Blue Native-PAGE samples from protein extracts.
It is advised to make master-mix of the 4x Native sample buffer, 5% Coomassie G-250 additive, and water. 
For 40 µL  of sample:
4x NativePAGETM sample buffer 20 µL 
5% Coomassie G-250 Additive 2 µL 
Distilled water 18 µL 
is added to make a total of 80 µL .

 

 

SECTION: Protein extraction
9. Make SDS-PAGE samples from the same sample protein extracts.
This is to ensure same samples are used for both blue native-PAGE and SDS-PAGE.
For 40 µL of sample:
3x SDS sample buffer 20 µL 
1 M DTT 8 µL 
Distilled water 12 µL 




SECTION: Protein extraction
9.1. Incubate samples at 70°C for 10 min .
After incubation, these samples are ready for SDS-PAGE.
SECTION: Protein extraction
2. Grind samples in 2 ml tubes using a tissue homogenizer (GenoGrinder), at 1200 rpm for 1 min . 
Samples should be kept frozen at all times, and put sample tubes back into liquid nitrogen immediately after sample grinding is complete.
 

SECTION: Protein extraction
3.  Open tube lids on ice, and add extraction buffer (see Materials).
The amount should be 50 µL .

SECTION: Protein extraction
4. Vortex for 10 seconds.15 s .

SECTION: Protein extraction
5. Incubate sample on rotation (must be kept cold) for 10 min .

SECTION: Protein extraction
6. Centrifuge at 4°C, 13,000 rpm, for 15 min .

SECTION: Protein extraction
7. Transfer supernatant to clean new tube, centrifuge again at 4°C, 13,000 rpm for 5 min .

 

SECTION: Protein extraction
8. Make Blue Native-PAGE samples from protein extracts.
It is advised to make master-mix of the 4x Native sample buffer, 5% Coomassie G-250 additive, and water. 
For 40 µL  of sample:
4x NativePAGETM sample buffer 20 µL 
5% Coomassie G-250 Additive 2 µL 
Distilled water 18 µL 
is added to make a total of 80 µL .

 

 

SECTION: Protein extraction
9. Make SDS-PAGE samples from the same sample protein extracts.
This is to ensure same samples are used for both blue native-PAGE and SDS-PAGE.
For 40 µL of sample:
3x SDS sample buffer 20 µL 
1 M DTT 8 µL 
Distilled water 12 µL 




SECTION: Protein extraction
9.1. Incubate samples at 70°C for 10 min .

SECTION: Protein extraction
3.  Open tube lids on ice, and add extraction buffer (see Materials).
The amount should be 50 µL , or 50 µL .

SECTION: Protein extraction
4. Vortex for 10 seconds.15 s .

SECTION: Protein extraction
5. Incubate sample on rotation (must be kept cold) for 10 min .

SECTION: Protein extraction
6. Centrifuge at 4°C, 13,000 rpm, for 15 min .

SECTION: Protein extraction
7. Transfer supernatant to clean new tube, centrifuge again at 4°C, 13,000 rpm for 5 min .

 

SECTION: Immunoprecipitation
2. Grind the N. benthamiana leaf samples using GenoGrinder, at 1200 rpm for 2 min  .

SECTION: Immunoprecipitation
3. Add extraction buffer, 1.7 mL to 2 ml tubes. 
This is to ensure buffer does not overflow.

SECTION: Immunoprecipitation
4. Vortex for 15 s .

SECTION: Immunoprecipitation
5. Incubate with rotation (in cold) for 10 min .

SECTION: Immunoprecipitation
7. Add 0.4 mL of new extraction buffer in clean tube, and transfer 1.6 mL supernatant from step 6.

SECTION: Immunoprecipitation
6. Centrifuge at 4°C, 13,000 rpm, for 15 min .

SECTION: Immunoprecipitation
10. Take 40 µL of protein extract from each sample, and make blue native-PAGE samples.

It is advised to make master-mix of the 4x Native sample buffer, 5% Coomassie G-250 additive, and water. 
For 40 µL  of sample:
4x NativePAGETM sample buffer 20 µL 
5% Coomassie G-250 Additive 2 µL 
Distilled water 18 µL 
is added to make a total of 80 µL .

 

 

SECTION: Immunoprecipitation
11. Take 40 µL of protein extract from each sample, and make SDS-PAGE samples.
This is to ensure same samples are used for both blue native-PAGE and SDS-PAGE, and can also be used as input of the following immunoprecipitation experiment.

For 40 µL of sample:
3x SDS sample buffer 20 µL 
1 M DTT 8 µL 
Distilled water 12 µL 




SECTION: Immunoprecipitation
11.1. Incubate samples at 70°C for 10 min .

SECTION: Immunoprecipitation
12. Equilibrate beads with extraction buffer.
Ideally, this step can be be done during centrifugation of protein extracts.
SECTION: Immunoprecipitation
8. Centrifuge at 4°C, 13,000 rpm, for 5 min .
SECTION: Immunoprecipitation
9. Transfer supernatant to clean tube.
SECTION: Immunoprecipitation
12.1. 40 µL bead slurry (50% slurry for Flag M2 beads, Sigma A2220) is taken and transferred to 2 ml tube.

 


SECTION: Immunoprecipitation
12.2. Add extraction buffer to fill the 2 ml tube, and invert the tubes several times so that beads are mixed well.
SECTION: Immunoprecipitation
12.3. Centrifuge at 5,000 rpm for 1 min , and remove remove supernatant.

SECTION: Immunoprecipitation
12.4. Repeat steps 12.2 and 12.3 two more times.
SECTION: Immunoprecipitation
12.5. Add 100 µL to the beads. 
If 120 μl bead slurry will be used for three samples, add 300 μl extraction buffer after removing final supernatant.
SECTION: Immunoprecipitation
12.6. In a new 2 ml tube, transfer 100 μl of bead + buffer from step 12.5.
The end of pipette tips should be cut off when pipetting beads to ensure accuracy.
SECTION: Immunoprecipitation
13. Add protein extracts from step 9 to tubes containing beads.
SECTION: Immunoprecipitation
14. Incubate on rotator (kept cold), for 120 min .

SECTION: Immunoprecipitation
15. Centrifuge samples at 4°C, 5,000 rpm for 1 min .

SECTION: Immunoprecipitation
16. Remove supernatant, add fresh wash buffer, and invert several times and make sure beads are floating.
SECTION: Immunoprecipitation
17. Repeat steps 15 and 16 for 2 more times with wash buffer.
SECTION: Immunoprecipitation
18. Repeat steps 15 and 16 for 2 more times with extraction buffer.
SECTION: Immunoprecipitation
19. Prepare elution buffer with 3x Flag peptide.
 

SECTION: Immunoprecipitation
20. To remove all of the buffer after the last wash, use gel loading tips or taper 200 μl tips with flat steel apparatus (e.g. back of spatula).
SECTION: Immunoprecipitation
21. Add 100 µL of elution buffer from step 19 to each sample.

 


SECTION: Immunoprecipitation
22. Incubate the beads on shaker (kept cold) for 60 min .

 


SECTION: Immunoprecipitation
23. Using tapered 200 μl tip, collect supernatants. 
After all supernatant is taken, cut off the end of the tip to remove any beads binding to the tip, and transfer eluate to new 1.5 ml tubes. 
SECTION: Immunoprecipitation
24. Take 40 µL of IP eluate from each sample, and make blue native-PAGE samples.

It is advised to make master-mix of the 4x Native sample buffer, 5% Coomassie G-250 additive, and water. 
For 40 µL  of sample:
4x NativePAGETM sample buffer 20 µL 
5% Coomassie G-250 Additive 2 µL 
Distilled water 18 µL 
is added to make a total of 80 µL .

 

 

SECTION: Immunoprecipitation
25. Take 40 µL of IP eluate from each sample, and make SDS-PAGE samples.
This is to ensure same samples are used for both blue native-PAGE and SDS-PAGE, and can also be used as input of the following immunoprecipitation experiment.

For 40 µL of sample:
3x SDS sample buffer 20 µL 
1 M DTT 8 µL 
Distilled water 12 µL 




SECTION: Immunoprecipitation
25.1. Incubate samples at 70°C for 10 min .
After incubation, samples are ready for SDS-PAGE.
SECTION: Immunoprecipitation
2. Grind samples to fine powder with mortar and pestle cooled in liquid nitrogen. 
Transfer finely ground powder into 50 ml tubes.
Samples should not thaw during this process.

SECTION: Immunoprecipitation
3. Add extraction buffer, 2 mL .

 

SECTION: Immunoprecipitation
4. Vortex until extract is homogeneous.
SECTION: Immunoprecipitation
5. Incubate with rotation (in cold) for 15 min .

SECTION: Immunoprecipitation
6. Centrifuge at 4°C, 5,000rpm, for 15 min .

SECTION: Immunoprecipitation
7. Pass supernatant through nylon mesh or microfilter, to remove any cell debris.
SECTION: Immunoprecipitation
8. Centrifuge at 4°C, 13,000rpm, for 20 min .
SECTION: Immunoprecipitation
9. Transfer supernatant to clean tube.
SECTION: Immunoprecipitation
10. Take 40 µL of protein extract from each sample, and make blue native-PAGE samples.

It is advised to make master-mix of the 4x Native sample buffer, 5% Coomassie G-250 additive, and water. 
For 40 µL  of sample:
4x NativePAGETM sample buffer 20 µL 
5% Coomassie G-250 Additive 2 µL 
Distilled water 18 µL 
is added to make a total of 80 µL .

 

 

SECTION: Immunoprecipitation
11. Take 40 µL of protein extract from each sample, and make SDS-PAGE samples.
This is to ensure same samples are used for both blue native-PAGE and SDS-PAGE, and can also be used as input of the following immunoprecipitation experiment.

For 40 µL of sample:
3x SDS sample buffer 20 µL 
1 M DTT 8 µL 
Distilled water 12 µL 




SECTION: Immunoprecipitation
11.1. Incubate samples at 70°C for 10 min .
After incubation, these samples are ready for SDS-PAGE.
SECTION: Immunoprecipitation
12. Equilibrate beads with extraction buffer.
Ideally, this step can be be done during centrifugation of protein extracts.
SECTION: Immunoprecipitation
12.1. 100 µL bead slurry (50% slurry for Flag M2 beads, Sigma A2220) is taken and transferred to 2 ml tube.

 


SECTION: Immunoprecipitation
12.2. Add extraction buffer to fill the 2 ml tube, and invert the tubes several times so that beads are mixed well.
SECTION: Immunoprecipitation
12.3. Centrifuge at 5,000 rpm for 1 min , and remove supernatant.

SECTION: Immunoprecipitation
12.4. Repeat steps 12.2 and 12.3 two more times.
SECTION: Immunoprecipitation
12.5. Add 200 µL to the beads. 
If 300 μl bead slurry will be used for three samples, add 600 μl extraction buffer after removing final supernatant.
SECTION: Immunoprecipitation
12.6. Transfer 220 μl of bead + buffer to protein extracts.
The end of pipette tips should be cut off when pipetting beads to ensure accuracy.
SECTION: Immunoprecipitation
13. Incubate on rotator (kept cold), for 120 min .

SECTION: Immunoprecipitation
14. Centrifuge samples at 4°C, 5,000 rpm for 1 min .

SECTION: Immunoprecipitation
15. Remove supernatant, add fresh wash buffer, and invert several times and make sure beads are floating.
SECTION: Immunoprecipitation
16. Repeat steps 15 and 16 for 2 more times with wash buffer.
SECTION: Immunoprecipitation
17. Repeat steps 15 and 16 for 2 more times with extraction buffer.
SECTION: Immunoprecipitation
18. Prepare elution buffer with 3x Flag peptide.
 

SECTION: Immunoprecipitation
19. To remove all of the buffer after the last wash, use gel loading tips or taper 200 μl tips with flat steel apparatus (e.g. back of spatula).
SECTION: Immunoprecipitation
20. Add 100 µL of elution buffer from step 19 to each sample.

 


SECTION: Immunoprecipitation
21. Incubate the beads on shaker (kept cold) for 60 min .

 


SECTION: Immunoprecipitation
22. Using tapered 200 μl tip, collect supernatants. 
After all supernatant is taken, cut off the end of the tip to remove any beads binding to the tip, and transfer eluate to new 1.5 ml tubes. 
SECTION: Immunoprecipitation
23. Take 40 µL of IP eluate from each sample, and make blue native-PAGE samples.

It is advised to make master-mix of the 4x Native sample buffer, 5% Coomassie G-250 additive, and water. 
For 40 µL  of sample:
4x NativePAGETM sample buffer 20 µL 
5% Coomassie G-250 Additive 2 µL 
Distilled water 18 µL 
is added to make a total of 80 µL .

 

 

SECTION: Immunoprecipitation
24. Take 40 µL of IP eluate from each sample, and make SDS-PAGE samples.
This is to ensure same samples are used for both blue native-PAGE and SDS-PAGE, and can also be used as input of the following immunoprecipitation experiment.

For 40 µL of sample:
3x SDS sample buffer 20 µL 
1 M DTT 8 µL 
Distilled water 12 µL 


SECTION: Immunoprecipitation
24.1. Incubate samples at 70°C for 10 min .
After incubation, sample is ready for SDS-PAGE.
SECTION: Protein extraction
8.1. Samples can be frozen in liquid nitrogen, and stored in -70°C until further use.
SECTION: Separation of samples by Blue Native-PAGE
11. Fill inner chamber with cold Cathode buffer (light) after removing comb. 

 

SECTION: Separation of samples by Blue Native-PAGE
12. Load samples. 10-15 μl (for 10-well gels). Add 5 μl of protein standard.
SECTION: Separation of samples by Blue Native-PAGE
13. Fill outer tank with cold Anode buffer.
SECTION: Separation of samples by Blue Native-PAGE
14. Gel running should be performed in cold room, or large refrigerator.

SECTION: Separation of samples by Blue Native-PAGE
15. Running condition: 150 V, 45 min (1/3 run into gel)
                                250 V, 45 min-1 h (until dye front reaches end of the gel)
Stop before dye front runs through the gel.

 

These gels can be used for protein transfer (next section), or separate strips can be used for 2 Dimensional blue-native/SDS-PAGE (see step case below).
SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
23. Assemble transfer cassette. Avoid forming air bubbles between gel and membrane. 

 

SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
24. Transfer is run with Transblot Turbo STANDARD method (20V constant, for 30 min ).

 

SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
25. After transfer is over, incubate the transferred membrane in 8% acetic acid for 15 min .

SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
26. Rinse membrane with clean water two times briefly.
SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
27. Place membrane on filter paper and let dry.
Up to 1 h in the fume hood, or overnight on bench.
SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
28. After membrane is completely dry, re-activate membrane with ethanol.
SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
29. As membrane is re-activated, Coomassie dye will wash out and markers will show up at this stage.
It is crucial to label all visible markers with pencil, as they will gradually disappear.
SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
29.1. For some proteins, the epitope may be buried within the protein complex making it difficult to detect with antibodies. 
To prevent this, incubate re-activated membrane with SDS running buffer (with up to 10 mM DTT) after re-activation 1 h to overnight. Wash thoroughly with TBS-T, 5 min 3 times.
SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
30. After membrane is re-activated and Coomassie dye is significantly washed away, decant the ethanol, and immediately add TBS-T. Wash membrane with TBS-T, 3 times for 2 min each.

 

SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
31. Add 5% skimmed milk in TBS-T to membrane, and block for 30 min .

SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
32. Decant blocking solution, and add antibody in 5% skimmed milk in TBS-T for 1 h to overnight.
SECTION: Protein transfer and immunoblotting of Blue Native-PAGE
33. If using HRP conjugated antibodies, membranes can be washed with TBS-T 3 times (5 min each) and will be ready for detecting chemiluminescent signal.
If using non-conjugated antibodies, wash with TBS-T 3 times (5 min each), incubate with secondary antibody (in 5% skimmed milk in TBS-T), then repeat washing step before preparing for chemiluminescent signal detection.
SECTION: Separation of samples by Blue Native-PAGE
16. Cut a strip of blue-native PAGE gel after electrophoresis is over, and carefully insert into 15 ml or 50 ml tube using gel holder or blunt/membrane forcep.
SECTION: Separation of samples by Blue Native-PAGE
17. Add 3X SDS sample buffer with 10 mM DTT into the tube, for 15 min .
SECTION: Separation of samples by Blue Native-PAGE
18. Rinse with water (at least three times).

 

SECTION: Separation of samples by Blue Native-PAGE
19. Prepare resolution gel of ideal percentage (for resolving Rpi-amr3 and NRC2, we used 8% gel). 

 

Resolution gel recipe for 8% gel:

 

SECTION: Separation of samples by Blue Native-PAGE
20. Insert the denatured gel strip onto the prepared resolution gel.
Add stacking gel buffer to stack gel strip on top of the resolution gel.
Ensure no air bubbles are stuck in between the gel strip and the resolution gel.

Stacking gel recipe:

 

 
SECTION: Separation of samples by Blue Native-PAGE
21. Run with identical conditions for SDS-PAGE.
SECTION: Separation of samples by blue native-PAGE
10. NativePAGE™ 3 to 12% pre-cast gel is used with XCellSureLock™ Mini-Cell system.

 

SECTION: Separation of samples by blue native-PAGE
11. Fill inner chamber with cold Cathode buffer (light) after removing comb. 

 

SECTION: Separation of samples by blue native-PAGE
12. Load samples. 10-15 μl (for 10-well gels). Add 5 μl of protein standard.
SECTION: Separation of samples by blue native-PAGE
13. Fill outer tank with cold Anode buffer.
SECTION: Separation of samples by blue native-PAGE
14. Gel running should be performed in cold room, or large refrigerator.

SECTION: Separation of samples by blue native-PAGE
15. Running condition: 150 V, 45 min (1/3 run into gel)
                                250 V, 45 min-1 h (until dye front reaches end of the gel)
Stop before dye front runs through the gel.

 

These gels can be used for protein transfer (next section), or separate strips can be used for 2 Dimensional blue-native/SDS-PAGE (see step case below).
SECTION: Separation of samples by blue-native PAGE
10. NativePAGE™ 3 to 12% pre-cast gel is used with XCellSureLock™ Mini-Cell system.

 

SECTION: Separation of samples by blue-native PAGE
11. Fill inner chamber with cold Cathode buffer (light) after removing comb. 

 

SECTION: Separation of samples by blue-native PAGE
12. Load samples. 10-15 μl (for 10-well gels). Add 5 μl of protein standard.
SECTION: Separation of samples by blue-native PAGE
13. Fill outer tank with cold Anode buffer.
SECTION: Separation of samples by blue-native PAGE
14. Gel running should be performed in cold room, or large refrigerator.

SECTION: Separation of samples by blue-native PAGE
15. Running condition: 150 V, 45 min (1/3 run into gel)
                                250 V, 45 min-1 h (until dye front reaches end of the gel)
Stop before dye front runs through the gel.

 

These gels can be used for protein transfer (next section), or separate strips can be used for 2 Dimensional blue-native/SDS-PAGE (see step case below).
SECTION: Separation of samples by blue-native PAGE
25. NativePAGE™ 3 to 12% pre-cast gel is used with XCellSureLock™ Mini-Cell system.

 

SECTION: Separation of samples by blue-native PAGE
26. Fill inner chamber with cold Cathode buffer (light) after removing comb. 

 

SECTION: Separation of samples by blue-native PAGE
27. Load samples. 10-15 μl (for 10-well gels). Add 5 μl of protein standard.
For IP eluates, up to 30 μl can be loaded.
SECTION: Separation of samples by blue-native PAGE
28. Fill outer tank with cold Anode buffer.
SECTION: Separation of samples by blue-native PAGE
29. Gel running should be performed in cold room, or large refrigerator.

SECTION: Separation of samples by blue-native PAGE
30. Running condition: 150 V, 45 min (1/3 run into gel)
                                250 V, 45 min-1 h (until dye front reaches end of the gel)
Stop before dye front runs through the gel.

 

These gels can be used for protein transfer (next section), or separate strips can be used for 2 Dimensional blue-native/SDS-PAGE (see step case below).
SECTION: Separation of samples by blue-native PAGE
26. NativePAGE™ 3 to 12% pre-cast gel is used with XCellSureLock™ Mini-Cell system.

 

SECTION: Separation of samples by blue-native PAGE
27. Fill inner chamber with cold Cathode buffer (light) after removing comb. 

 

SECTION: Separation of samples by blue-native PAGE
28. Load samples. 10-15 μl (for 10-well gels). Add 5 μl of protein standard.
For IP eluates, up to 30 μl can be loaded.
SECTION: Separation of samples by blue-native PAGE
29. Fill outer tank with cold Anode buffer.
SECTION: Separation of samples by blue-native PAGE
30. Gel running should be performed in cold room, or large refrigerator.

SECTION: Separation of samples by blue-native PAGE
31. Running condition: 150 V, 45 min (1/3 run into gel)
                                250 V, 45 min-1 h (until dye front reaches end of the gel)
Stop before dye front runs through the gel.

 

These gels can be used for protein transfer (next section), or separate strips can be used for 2 Dimensional blue-native/SDS-PAGE (see step case below).
SECTION: Protein transfer and immunoblotting of blue native-PAGE
16. After gels are run until dye fron reaches end of gel, gel cassette is pry open using cassette opening lever/gel knife.
Lift open all three sides of the cassette, and carefully cut both ends of the gel (side with wells and the opposite side where the dye front is).
SECTION: Protein transfer and immunoblotting of blue native-PAGE
17. Assemble transfer cassette. Avoid forming air bubbles between gel and membrane. 

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
18. Transfer is run with Transblot Turbo STANDARD method (20V constant, for 30 min ).

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
19. After transfer is over, incubate the transferred membrane in 8% acetic acid for 15 min .

SECTION: Protein transfer and immunoblotting of blue native-PAGE
20. Rinse membrane with clean water two times briefly.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
21. Place membrane on filter paper and let dry.
Up to 1 h in the fume hood, or overnight on bench.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
22. After membrane is completely dry, re-activate membrane with ethanol.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
23. As membrane is re-activated, Coomassie dye will wash out and markers will show up at this stage.
It is crucial to label all visible markers with pencil, as they will gradually disappear.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
23.1. For some proteins, the epitope may be buried within the protein complex making it difficult to detect with antibodies. 
To prevent this, incubate re-activated membrane with SDS running buffer (with up to 10 mM DTT) after re-activation 1 h to overnight. Wash thoroughly with TBS-T, 5 min 3 times.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
24. After membrane is re-activated and Coomassie dye is significantly washed away, decant the ethanol, and immediately add TBS-T. Wash membrane with TBS-T, 3 times for 2 min each.

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
25. Add 5% skimmed milk in TBS-T to membrane, and block for 30 min .

SECTION: Protein transfer and immunoblotting of blue native-PAGE
26. Decant blocking solution, and add antibody in 5% skimmed milk in TBS-T for 1 h to overnight.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
27. If using HRP conjugated antibodies, membranes can be washed with TBS-T 3 times (5 min each) and will be ready for detecting chemiluminescent signal.
If using non-conjugated antibodies, wash with TBS-T 3 times (5 min each), incubate with secondary antibody (in 5% skimmed milk in TBS-T), then repeat washing step before preparing for chemiluminescent signal detection.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
16. After gels are run until dye fron reaches end of gel, gel cassette is pry open using cassette opening lever/gel knife.
Lift open all three sides of the cassette, and carefully cut both ends of the gel (side with wells and the opposite side where the dye front is).
SECTION: Protein transfer and immunoblotting of blue native-PAGE
17. Assemble transfer cassette. Avoid forming air bubbles between gel and membrane. 

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
18. Transfer is run with Transblot Turbo STANDARD method (20V constant, for 30 min ).

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
19. After transfer is over, incubate the transferred membrane in 8% acetic acid for 15 min .

SECTION: Protein transfer and immunoblotting of blue native-PAGE
20. Rinse membrane with clean water two times briefly.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
21. Place membrane on filter paper and let dry.
Up to 1 h in the fume hood, or overnight on bench.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
22. After membrane is completely dry, re-activate membrane with ethanol.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
23. As membrane is re-activated, Coomassie dye will wash out and markers will show up at this stage.
It is crucial to label all visible markers with pencil, as they will gradually disappear.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
23.1. For some proteins, the epitope may be buried within the protein complex making it difficult to detect with antibodies. 
To prevent this, incubate re-activated membrane with SDS running buffer (with up to 10 mM DTT) after re-activation 1 h to overnight. Wash thoroughly with TBS-T, 5 min 3 times.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
24. After membrane is re-activated and Coomassie dye is significantly washed away, decant the ethanol, and immediately add TBS-T. Wash membrane with TBS-T, 3 times for 2 min each.

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
25. Add 5% skimmed milk in TBS-T to membrane, and block for 30 min .

SECTION: Protein transfer and immunoblotting of blue native-PAGE
26. Decant blocking solution, and add antibody in 5% skimmed milk in TBS-T for 1 h to overnight.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
27. If using HRP conjugated antibodies, membranes can be washed with TBS-T 3 times (5 min each) and will be ready for detecting chemiluminescent signal.
If using non-conjugated antibodies, wash with TBS-T 3 times (5 min each), incubate with secondary antibody (in 5% skimmed milk in TBS-T), then repeat washing step before preparing for chemiluminescent signal detection.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
31. After gels are run until dye fron reaches end of gel, gel cassette is pry open using cassette opening lever/gel knife.
Lift open all three sides of the cassette, and carefully cut both ends of the gel (side with wells and the opposite side where the dye front is).
SECTION: Protein transfer and immunoblotting of blue native-PAGE
32. Assemble transfer cassette. Avoid forming air bubbles between gel and membrane. 

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
33. Transfer is run with Transblot Turbo STANDARD method (20V constant, for 30 min ).

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
34. After transfer is over, incubate the transferred membrane in 8% acetic acid for 15 min .

SECTION: Protein transfer and immunoblotting of blue native-PAGE
35. Rinse membrane with clean water two times briefly.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
36. Place membrane on filter paper and let dry.
Up to 1 h in the fume hood, or overnight on bench.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
37. After membrane is completely dry, re-activate membrane with ethanol.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
38. As membrane is re-activated, Coomassie dye will wash out and markers will show up at this stage.
It is crucial to label all visible markers with pencil, as they will gradually disappear.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
38.1. (Optional)
For some proteins, the epitope may be buried within the protein complex making it difficult to detect with antibodies. 
To prevent this, incubate re-activated membrane with SDS running buffer (with up to 10 mM DTT) after re-activation 1 h to overnight. Wash thoroughly with TBS-T, 5 min 3 times.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
39. After membrane is re-activated and Coomassie dye is significantly washed away, decant the ethanol, and immediately add TBS-T. Wash membrane with TBS-T, 3 times for 2 min each.

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
40. Add 5% skimmed milk in TBS-T to membrane, and block for 30 min .

SECTION: Protein transfer and immunoblotting of blue native-PAGE
41. Decant blocking solution, and add antibody in 5% skimmed milk in TBS-T for 1 h to overnight.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
42. If using HRP conjugated antibodies, membranes can be washed with TBS-T 3 times (5 min each) and will be ready for detecting chemiluminescent signal.
If using non-conjugated antibodies, wash with TBS-T 3 times (5 min each), incubate with secondary antibody (in 5% skimmed milk in TBS-T), then repeat washing step before preparing for chemiluminescent signal detection.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
32. After gels are run until dye front reaches end of gel, gel cassette is pry open using cassette opening lever/gel knife.
Lift open all three sides of the cassette, and carefully cut both ends of the gel (side with wells and the opposite side where the dye front is).
SECTION: Protein transfer and immunoblotting of blue native-PAGE
33. Assemble transfer cassette. Avoid forming air bubbles between gel and membrane. 

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
34. Transfer is run with Transblot Turbo STANDARD method (20V constant, for 30 min ).

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
35. After transfer is over, incubate the transferred membrane in 8% acetic acid for 15 min .

SECTION: Protein transfer and immunoblotting of blue native-PAGE
36. Rinse membrane with clean water two times briefly.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
37. Place membrane on filter paper and let dry.
Up to 1 h in the fume hood, or overnight on bench.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
38. After membrane is completely dry, re-activate membrane with ethanol.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
39. As membrane is re-activated, Coomassie dye will wash out and markers will show up at this stage.
It is crucial to label all visible markers with pencil, as they will gradually disappear.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
39.1. For some proteins, the epitope may be buried within the protein complex making it difficult to detect with antibodies. 
To prevent this, incubate re-activated membrane with SDS running buffer (with up to 10 mM DTT) after re-activation 1 h to overnight. Wash thoroughly with TBS-T, 5 min 3 times.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
40. After membrane is re-activated and Coomassie dye is significantly washed away, decant the ethanol, and immediately add TBS-T. Wash membrane with TBS-T, 3 times for 2 min each.

 

SECTION: Protein transfer and immunoblotting of blue native-PAGE
41. Add 5% skimmed milk in TBS-T to membrane, and block for 30 min .

SECTION: Protein transfer and immunoblotting of blue native-PAGE
42. Decant blocking solution, and add antibody in 5% skimmed milk in TBS-T for 1 h to overnight.
SECTION: Protein transfer and immunoblotting of blue native-PAGE
43. If using HRP conjugated antibodies, membranes can be washed with TBS-T 3 times (5 min each) and will be ready for detecting chemiluminescent signal.
If using non-conjugated antibodies, wash with TBS-T 3 times (5 min each), incubate with secondary antibody (in 5% skimmed milk in TBS-T), then repeat washing step before preparing for chemiluminescent signal detection.
SECTION: Protein extraction
8.1. Samples can be snap-frozen and kept in -80°C until further use.
SECTION: Protein extraction
2. Grind samples in 2 ml tubes using a tissue homogenizer (GenoGrinder), at 1200 rpm for 1 min . 
Samples should be kept frozen at all times, and put sample tubes back into liquid nitrogen immediately after sample grinding is complete.
 

SECTION: Protein extraction
8. Make Blue Native-PAGE samples from protein extracts.
It is advised to make master-mix of the 4x Native sample buffer, 5% Coomassie G-250 additive, and water. 
For 40 µL  of sample:
4x NativePAGETM sample buffer 20 µL 
5% Coomassie G-250 Additive 2 µL 
Distilled water 18 µL 
is added to make a total of 80 µL .

 

 

SECTION: Protein extraction
8.1. Samples can be frozen in liquid nitrogen, and stored in -70°C until further use.
SECTION: Protein extraction
9. Make SDS-PAGE samples from the same sample protein extracts.
This is to ensure same samples are used for both blue native-PAGE and SDS-PAGE.
For 40 µL of sample:
3x SDS sample buffer 20 µL 
1 M DTT 8 µL 
Distilled water 12 µL 




SECTION: Protein extraction
9.1. Incubate samples at 70°C for 10 min .
After incubation, these samples are ready for SDS-PAGE.
SECTION: Immunoprecipitation
10.1. Samples can be frozen in liquid nitrogen, and stored in -70°C until further use.
SECTION: Immunoprecipitation
23.1. Immediately freeze blue native-PAGE samples in liquid nitrogen and store samples in -70°C freezer until further use.
SECTION: Immunoprecipitation
10.1. Samples are frozen in liquid nitrogen, and stored in -70°C until further use.
SECTION: Immunoprecipitation
24.1. Samples can be frozen in liquid nitrogen, and stored in -70°C until further use.