Western CJF

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Notebook | Protocols

Contents

BRADFORD ASSAY

Materials

  • Protein assay dye 5x (BioRad)
  • 2ml microcentrifuge tubes
  • 96 well plate flatebottom plate
  • Plate reader
  • Distilled H2O
  • 2mg/mL BSA

Protein Assay (Bradford)

  1. In 96 well plate, carefully pipette 200μL protein assay dye 1x (BioRad) in each well. Protein assay comes as 5x concentrate, dilute with distilled H20.
  2. Standard: Prepare 7 standards via serial dilution. Starting with 30uL distilled H2O and 30μL BSA standard [2mg/mL]. To each remaining standard, add 30 μL of the previous sample. The final sample will have 60μL
  3. Sample: To 27 μL Distilled H2O, add 3 μL sample. If sample is suspected to be very concentrated, test another dilution of 3uL in 57uL distilled H2O
  4. Add 10μL sample to wells in duplicate, mix well by pipette.
  5. Incubate at room temperature ~5 min.
  6. Running the sample: Choose old plate as template and modify to fit current samples. Ensure that Standards and Unknown concentrations are correct (Standard is Serial dilution, 2x, X1 is most concentrated; Unknown is 10x dilution or 20x dilution) Save as a new file. Open Endpoint program and pick presaved plate file. Change reports to include raw data and standards. Change wavelength to read A593.
  7. Export Concentrations to excel, remember to start export at cell 1, and email to self.



Bio-Rad Based Western

Materials

  • Protein sample
  • Protein Ladder
  • milipore water
  • NuPAGE® LDS Sample Buffer
  • NuPAGE® Novex Bis-Tris Gel: Gel Thickness: 1.0 mm, Gel Sze: 10 well gel: 8 cm x 8 cm, Load Volume: 25 μl
  • NuPAGE® Novex Bis-Tris Gel: Gel Thickness: 1.0 mm, Gel Sze: 15 well gel: 8 cm x 8 cm, Load Volume: 18 μl
  • NuPAGE® SDS Running Buffer
  • Electrophoresis module
  • Blotting membranes
  • Filter paper
  • Methanol (if using PVDF)
  • XCell II. Blot module
  • NuPAGE® Transfer Buffer
  • Beta-mercapenethanol

Procedure

  • Running the Gel
  1. Wear protective gloves when handling gels and solutions
  2. Determine amount of sample needed for desired µg of protein in each electrophoresis sample. 25 μg proteins
  3. Prepare reducing sample
  4. Sample (depends on Bradford assay) μl
  5. NuPAGE LDS Sample Buffer (4X) 3-5μl/sample
  6. Beta-Mercapoethanol (10X)
  7. Heat the sample for denaturing electrophoresis (reduced or non-reduced) at 100°C for 10 minutes for optimal results.
  8. Prepare 1000 ml of 1x NuPAGE® SDS Running Buffer using NuPAGE® SDS Running Buffer(20X) as follows: Running Buffer:
    1. NuPAGE® SDS Running Buffer (20X MES) 50 ml
    2. Deionized Water 950 ml
    3. Total Volume 1000 ml
    4. Mix thoroughly and set aside 800 ml of the 1X NuPAGE® SDS Running Buffer for use in the lower (Outer) Buffer Chamber of the XCell SureLock Mini-Cell
  9. Remove the NuPAGE® Gel from the pouch
  10. Rinse the gel cassette with deionized water. Peel off the tape from the bottom of the cassette.
  11. In one smooth motion, gently pull the comb out of the cassette
  12. Rinse the sample wells with 1X NuPAGE® SDS Running Buffer. Invert the gel and shake to remove the buffer. Repeat two more times.
  13. Orient the two gels in the Mini-Cell such that the notched well-side of the cassette faces inwards toward the Buffer Core. Seat the gels on the bottom of the Mini-Cell and lock into place with the Gel Tension Wedge.
  14. If you are using only one gel, the plastic Buffer Dam replaces the second gel cassette
  15. Fill the Upper Buffer Chamber with a small amount of the running buffer to check for tightness of seal. If you detect a leak from Upper to Lower Buffer Chamber, discard the buffer, reseal the chamber, and refill.
  16. Once the seal is tight, fill the Upper Buffer Chamber (inner) with the appropriate 1X running buffer. The buffer level must exceed the level of the wells.
  17. Load an appropriate volume of sample at the desired protein concentration onto the gel
  18. Load appropriate protein molecular weight markers
  19. Fill the Lower (outer) Buffer Chamber with 600 ml of 1X NuPAGE® SDS Running Buffer
  20. Run the gel at 200 V constant. Run for 65 minutes. (Check for bubbles emerging from solution to see if electrophoresis is running.)


  • Removing the Gel after Electrophorsis
  1. After electrophoresis is complete, shut off the power, disconnect electrodes, and remove gel(s) from the XCell SureLock. Mini-Cell
  2. Separate each of the three bonded sides of the cassette by inserting the Gel Knife into the gap between the cassette’s two plates. The notched (well) side of the cassette should face up.
  3. Push down gently on the knife handle to separate the plates. Repeat on each side of the cassette until the plates are completely separated
  4. Carefully remove and discard the top plate, allowing the gel to remain on the bottom (slotted) plate


Western Blotting

  1. Prepare 1000 ml of 1X NuPAGE® Transfer Buffer using the NuPAGE® Transfer Buffer (20X) as follows:Transfer Buffer:
  2. NuPAGE® Transfer Buffer (20X) 50 ml
  3. Methanol 100 ml
  4. Deionized Water 850 ml
  5. Total Volume 1000 ml
  6. Use about 700 ml of 1X NuPAGE® Transfer Buffer to soak the pads until saturated. Remove the air bubbles by squeezing the pads while they are submerged in buffer. Removing the air bubbles is essential as they can block the transfer of biomolecules if they are not removed.
  7. Cut selected transfer membrane and filter paper to the dimensions of the gel or use Novex® pre-cut membrane/filter paper sandwich
  8. Pre-wet PVDF membrane for 30 seconds in methanol. Then place in a shallow dish with 50 ml of 1X NuPAGE® Transfer Buffer for several minutes. Marked THP1 membrane with wet-marker on side facing membrane.
  9. Soak the filter paper briefly in 1X NuPAGE® Transfer Buffer immediately prior to use.
  10. Use the gel immediately following the run. Do not soak the gel in transfer buffer
  11. After opening the gel cassette remove wells with the Gel Knife
  12. Place a piece of pre-soaked filter paper on top of the gel, and lay just above the slot in the bottom of the cassette, leaving the foot of the gel uncovered. Keep the filter paper saturated with the transfer buffer and remove all trapped air bubbles by gently rolling over the surface using a glass pipette as a roller
  13. Turn the plate over so the gel and filter paper are facing downwards over a gloved hand or clean flat surface
  14. Use the Gel Knife to push the foot out of the slot in the plate and the gel will fall off
  15. When the gel is on a flat surface, cut the foot off the gel with the gel knife
  16. Wet the surface of the gel with transfer buffer and position the pre-soaked transfer membrane on the gel, ensuring all air bubbles have been removed
  17. Place another pre-soaked anode filter paper on top of the membrane. Remove any trapped air bubbles
  18. Place two soaked blotting pads into the cathode (-) core of the blot module. Placed two soaked blotting pads into the cathode. Placed 15 well membrane towards back. Placed two soaked blotting pads. 10 well membrane. Placed two soaked blotting pads. The gel is closest to the cathode core .
  19. Add enough pre-soaked blotting pads to rise to 0.5 cm over rim of cathode core. Place the anode (+) core on top of the pads. The gel/membrane assembly should be held securely between the two halves of the blot module ensuring complete contact of all components
  20. Position the gel/membrane assembly and blotting pads in the cathode core of the XCell II. Blot Module to fit horizontally across the bottom of the unit. There should be a gap of approximately 1 cm at the top of the electrodes when the pads and assembly are in place
  21. Hold the blot module together firmly and slide it into the guide rails on the lower buffer chamber. The blot module will only fit into the unit one way, so the (+) sign can be seen in the upper left hand corner of the blot module. Properly placed, the inverted gold post on the right hand side of the blot module will fit into the hole next to the upright gold post on the right side of the lower buffer chamber
  22. Place the Gel Tension Wedge so that its vertical face is against the blot module. Lock the Gel Tension Wedge by pulling the lever forward
  23. Fill the blot module with 1X NuPAGE® Transfer Buffer until the gel/membrane assembly is covered in this buffer. Do not fill all the way to the top as this will only generate extra conductivity and heat
  24. Fill the Outer Buffer Chamber with deionized water by pouring approximately 650 ml in the gap between the front of the blot module and the front of the lower buffer chamber. The water level should reach approximately 2 cm from the top of the lower buffer chamber. This serves to dissipate heat produced during the run
  25. Place the lid on top of the unit
  26. With the power turned on, plug the red and black leads into the power supply. Run the transfer at 30 Volts with an expected current of 170mA for 65 minutes

Immunoblot Assay

  1. Rinse membrane twice with water
  2. Block membrane 1hr at room temperature with agitation in freshly prepared 5% nonfat dry milk (Catalog 20-200) in TBS (TBS=PBS-T: 1:1000 TWEEN in PBS) 20mL solution per PVDF.
  3. Rinse with TBS x1 and shake at RT with agitation for 5minutes x2.
  4. Incubate membrane overnight at 4C with agitation and Primary antibody, diluted 1:1000 in freshly prepared TBS. 5mL total volume per membrane: pYSTAT3(705)
  5. Wash membrane twice (5-10min each) with PBS-T
  6. Incubate membrane for 1hr at room temperature with agitation in anti-rabbit conjugate (1.5:10,000 dilution in PBS-T)
  7. Wash membrane 2x (5-10min each) with PBS-T

Chemiluminescence Detection

  1. Mix 0.8 mL of Bio-Rad Detection Reagent 1 and 2 (0.2 mL of each, depending on size of membrane), with a pipette.
  2. Pour over transfer membrane and leave on for one minute.
  3. Dry off with Kimwipe

Stripping Membrane

  • Materials
  • 62.5 mM Tris
  • 2% SDS
  • 0.1 M beta-mercapetanol
  1. Prepare solution in 50mL falcon (3.125mL Tris, 350uL beta-merc, 10mL of 10% SDS, Remaining water)
  2. Incubate at 50C while spinning for 1hrminutes
  3. Wash with PBS-T x3 for 5minutes with agitiation
  4. Return to "Immunoblot Assay" step

Immunoblot Assay

  1. Rinse membrane twice with water
  2. Block membrane O/N at 4C with agitation in freshly prepared 5% nonfat dry milk (Catalog 20-200) in PBS-T (PBS-T: 1:1000 TWEEN in PBS) 20mL solution per PVDF.
  3. Rinse with PBS-T x1 and shake at RT with agitation for 5 minutes x2.
  4. Incubate membrane overnight at 4C with agitation and Primary antibody, diluted (generally 1:1000) in freshly prepared PBS-T. 5 mL total volume per membrane:
  5. Wash membrane twice (5-10min each) with PBS-T
  6. Incubate membrane for 1hr at room temperature with agitation in anti-rabbit conjugate (1:10000 dilution in PBS-T)
  7. Wash membrane 3x (5-10min each) with PBS-T

Western

  • Probing for pYSTAT3: tSTAT3 (90kb), pY416SRC:tSRC (60kd), SOCS3 (23kd)
Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Kodak Based Western

Materials

  • Protein sample
  • Protein Ladder
  • milipore water
  • NuPAGE® LDS Sample Buffer
  • NuPAGE® Novex Bis-Tris Gel: Gel Thickness: 1.0 mm, Gel Sze: 10 well gel: 8 cm x 8 cm, Load Volume: 25 μl
  • NuPAGE® Novex Bis-Tris Gel: Gel Thickness: 1.0 mm, Gel Sze: 15 well gel: 8 cm x 8 cm, Load Volume: 18 μl
  • NuPAGE® SDS Running Buffer
  • Electrophoresis module
  • Blotting membranes
  • Filter paper
  • Methanol (if using PVDF)
  • XCell II. Blot module
  • NuPAGE® Transfer Buffer
  • Beta-mercapenethanol

Procedure

  • Running the Gel
  1. Wear protective gloves when handling gels and solutions
  2. Determine amount of sample needed for desired µg of protein in each electrophoresis sample. 25 μg proteins
  3. Prepare reducing sample
  4. Sample (depends on Bradford assay) μl
  5. NuPAGE LDS Sample Buffer (4X) 3-5μl/sample
  6. Beta-Mercapoethanol (10X)
  7. Heat the sample for denaturing electrophoresis (reduced or non-reduced) at 100°C for 10 minutes for optimal results.
  8. Prepare 1000 ml of 1x NuPAGE® SDS Running Buffer using NuPAGE® SDS Running Buffer(20X) as follows: Running Buffer:
    1. NuPAGE® SDS Running Buffer (20X MES) 50 ml
    2. Deionized Water 950 ml
    3. Total Volume 1000 ml
    4. Mix thoroughly and set aside 800 ml of the 1X NuPAGE® SDS Running Buffer for use in the lower (Outer) Buffer Chamber of the XCell SureLock Mini-Cell
  9. Remove the NuPAGE® Gel from the pouch
  10. Rinse the gel cassette with deionized water. Peel off the tape from the bottom of the cassette.
  11. In one smooth motion, gently pull the comb out of the cassette
  12. Rinse the sample wells with 1X NuPAGE® SDS Running Buffer. Invert the gel and shake to remove the buffer. Repeat two more times.
  13. Orient the two gels in the Mini-Cell such that the notched well-side of the cassette faces inwards toward the Buffer Core. Seat the gels on the bottom of the Mini-Cell and lock into place with the Gel Tension Wedge.
  14. If you are using only one gel, the plastic Buffer Dam replaces the second gel cassette
  15. Fill the Upper Buffer Chamber with a small amount of the running buffer to check for tightness of seal. If you detect a leak from Upper to Lower Buffer Chamber, discard the buffer, reseal the chamber, and refill.
  16. Once the seal is tight, fill the Upper Buffer Chamber (inner) with the appropriate 1X running buffer. The buffer level must exceed the level of the wells.
  17. Load an appropriate volume of sample at the desired protein concentration onto the gel
  18. Load appropriate protein molecular weight markers
  19. Fill the Lower (outer) Buffer Chamber with 600 ml of 1X NuPAGE® SDS Running Buffer
  20. Run the gel at 200 V constant. Run for 80 minutes. (Check for bubbles emerging from solution to see if electrophoresis is running.)


  • Removing the Gel after Electrophorsis
  1. After electrophoresis is complete, shut off the power, disconnect electrodes, and remove gel(s) from the XCell SureLock. Mini-Cell
  2. Separate each of the three bonded sides of the cassette by inserting the Gel Knife into the gap between the cassette’s two plates. The notched (well) side of the cassette should face up.
  3. Push down gently on the knife handle to separate the plates. Repeat on each side of the cassette until the plates are completely separated
  4. Carefully remove and discard the top plate, allowing the gel to remain on the bottom (slotted) plate


Western Blotting

  1. Prepare 1000 ml of 1X NuPAGE® Transfer Buffer using the NuPAGE® Transfer Buffer (20X) as follows:Transfer Buffer:
  2. NuPAGE® Transfer Buffer (20X) 50 ml
  3. Methanol 100 ml
  4. Deionized Water 850 ml
  5. Total Volume 1000 ml
  6. Use about 700 ml of 1X NuPAGE® Transfer Buffer to soak the pads until saturated. Remove the air bubbles by squeezing the pads while they are submerged in buffer. Removing the air bubbles is essential as they can block the transfer of biomolecules if they are not removed.
  7. Cut selected transfer membrane and filter paper to the dimensions of the gel or use Novex® pre-cut membrane/filter paper sandwich
  8. Pre-wet PVDF membrane for 30 seconds in methanol. Then place in a shallow dish with 50 ml of 1X NuPAGE® Transfer Buffer for several minutes. Marked THP1 membrane with wet-marker on side facing membrane.
  9. Soak the filter paper briefly in 1X NuPAGE® Transfer Buffer immediately prior to use.
  10. Use the gel immediately following the run. Do not soak the gel in transfer buffer
  11. After opening the gel cassette remove wells with the Gel Knife
  12. Place a piece of pre-soaked filter paper on top of the gel, and lay just above the slot in the bottom of the cassette, leaving the foot of the gel uncovered. Keep the filter paper saturated with the transfer buffer and remove all trapped air bubbles by gently rolling over the surface using a glass pipette as a roller
  13. Turn the plate over so the gel and filter paper are facing downwards over a gloved hand or clean flat surface
  14. Use the Gel Knife to push the foot out of the slot in the plate and the gel will fall off
  15. When the gel is on a flat surface, cut the foot off the gel with the gel knife
  16. Wet the surface of the gel with transfer buffer and position the pre-soaked transfer membrane on the gel, ensuring all air bubbles have been removed
  17. Place another pre-soaked anode filter paper on top of the membrane. Remove any trapped air bubbles
  18. Place two soaked blotting pads into the cathode (-) core of the blot module. Placed two soaked blotting pads into the cathode. Placed 15 well membrane towards back. Placed two soaked blotting pads. 10 well membrane. Placed two soaked blotting pads. The gel is closest to the cathode core .
  19. Add enough pre-soaked blotting pads to rise to 0.5 cm over rim of cathode core. Place the anode (+) core on top of the pads. The gel/membrane assembly should be held securely between the two halves of the blot module ensuring complete contact of all components
  20. Position the gel/membrane assembly and blotting pads in the cathode core of the XCell II. Blot Module to fit horizontally across the bottom of the unit. There should be a gap of approximately 1 cm at the top of the electrodes when the pads and assembly are in place
  21. Hold the blot module together firmly and slide it into the guide rails on the lower buffer chamber. The blot module will only fit into the unit one way, so the (+) sign can be seen in the upper left hand corner of the blot module. Properly placed, the inverted gold post on the right hand side of the blot module will fit into the hole next to the upright gold post on the right side of the lower buffer chamber
  22. Place the Gel Tension Wedge so that its vertical face is against the blot module. Lock the Gel Tension Wedge by pulling the lever forward
  23. Fill the blot module with 1X NuPAGE® Transfer Buffer until the gel/membrane assembly is covered in this buffer. Do not fill all the way to the top as this will only generate extra conductivity and heat
  24. Fill the Outer Buffer Chamber with deionized water by pouring approximately 650 ml in the gap between the front of the blot module and the front of the lower buffer chamber. The water level should reach approximately 2 cm from the top of the lower buffer chamber. This serves to dissipate heat produced during the run
  25. Place the lid on top of the unit
  26. With the power turned on, plug the red and black leads into the power supply. Run the transfer at 30 Volts with an expected current of 170mA for 80 minutes

Immunoblot Assay

  1. Rinse membrane twice with water
  2. Block membrane 1hr at room temperature with agitation in freshly prepared 5% nonfat dry milk (Catalog 20-200) in TBS (TBS=PBS-T: 1:1000 TWEEN in PBS) 20mL solution per PVDF.
  3. Rinse with TBS x1 and shake at RT with agitation for 5minutes x2.
  4. Incubate membrane overnight at 4C with agitation and Primary antibody, diluted 1:1000 in freshly prepared TBS. 10mL total volume per membrane: pYSTAT3(705)
  5. Wash membrane twice (5-10min each) with TBS
  6. Incubate membrane for 1hr at room temperation with agitation in anti-rabbit conjugate (3:10,000 dilution in TBS)
  7. Wash membrane 2x (5-10min each) with TBS

Chemiluminescence Detection

  1. Mix 0.8 mL of Bio-Rad Detection Reagent 1 and 2 (0.4 mL of each), with a pipette.
  2. Pour over transfer membrane and leave on for one minute.
  3. Dry off with Kimwipe
  4. Bring to the Bio-Rad and make sure the door is tightly closed (no light seeping through), before beginning
  5. Take out Kodak scientific imaging film and cut in half. Put the other half back in the box.
  6. Put Kodak film over transfer membrane in cassette and close cassette. Leave for one minute. (Note: Do not expose film to light. Keep lights off until film is in imaging machine)
  7. Put film into imaging machine to process
  8. If nothing shows, put the film back into the cassette and leave it in for another 30 minutes. Check back to see if results show.

Stripping Membrane

  • Materials
  • 62.5 mM Tris
  • 2% SDS
  • 0.1 M beta-mercapetanol
  1. Prepare solution in 50mL falcon (3.125mL Tris, 350uL beta-merc, 10mL of 10% SDS, Remaining water)
  2. Incubate at 50C while spinning for 1hrminutes
  3. Wash with TBS x3 for 5minutes with agitiation
  4. Return to "Immunoblot Assay" step

Immunoblot Assay

  1. Rinse membrane twice with water
  2. Block membrane O/N at 4C with agitation in freshly prepared 5% nonfat dry milk (Catalog 20-200) in TBS (TBS=PBS-T: 1:1000 TWEEN in PBS) 20mL solution per PVDF.
  3. Rinse with TBS x1 and shake at RT with agitation for 5minutes x2.
  4. Incubate membrane 3 days at 4C with agitation and Primary antibody, diluted (generally 1:1000) in freshly prepared TBS. 10mL total volume per membrane:
  5. Wash membrane twice (5-10min each) with TBS
  6. Incubate membrane for 1hr at room temperation with agitation in anti-rabbit conjugate (1:10000 dilution in TBS)
  7. Wash membrane 3x (5-10min each) with TBS
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