1
Western Blotting Procedure
Isolating Proteins
1.
2.
3.
4.
ONLY IF CELLS ARE DEAD: Collect media
Wash once or twice with PBS. ONLY IF CELLS ARE DEAD: Collect media
IMPORTANT: If you do not wash well, the trypsin won’t detach the cells properly
Add trypsin and incubate for 5-10 min
a. ALL cells should be detached. In case trypsin fails to detach cells, collect trypsin
and add more fresh trypsin for another 5 min.
5. Inactivate trypsin with twice as much media. Collect everything making sure you wash
all cells in the plate and transfer them to the same centrifuge tube.
6. Transfer cells to ice
7. Centrifuge cells at 300 g 4 oC for 5 min.
8. Get rid of supernatant
9. Add 50-100ul (usually 100ul per 100mm dish) RIPA buffer (DO NOT FORGET TO ADD THE
PROTEASE/PHOSPHATASE INHIBITORS).
10. Homogenize (sonicate 10% amplitude) samples for 5 seconds. Remember to clean-up
homogeneizer before and after every sample. Do not touch the bottom of the tube
while homogenizing. USE HEADPHONES
a. Sonicator is turned on via a switch in the underside of the front of the machine
b. Keep your samples on ice
c. Wait after turning on until a BEEP sound is heard
d. Press Start
e. Switch off when done
11. If necessary, transfer cells to a microcentrifuge (1.5 ml tube) and keep on ice.
12. Store samples at -80 oC
RIPA buffer (250 ml)
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2.1915g NaCl
2.5 ml Triton X-100 (1% final)
1.25 g Sodium deoxycholate (0.5 % final)
0.25 g SDS (0.1 % final)
25 ml Tris base 0.5M (50 mM final) pH – 8
o Stock 0.5M pH – 8 (15.14g in 250 ml)
 Dissolve and make up to 250 ml with water
PRIOR TO USE: Add Halt protease inhibitors 100X. 1ul per 100ul RIPA
2
Quantifying Proteins Protocol
1.
2.
3.
4.
5.
Turn on the centrifuge
Take proteins out of the freezer and place them in an ice bucket
Once thawed, centrifuge the protein at 10,000 rpm (min^-1) 4°C for 5 minutes.
Place the proteins back in the ice after centrifugation
Mix Reagent A&B form BCA Pierce kit, according to the 50:1 ratio in a 15ml tube
Example:
Reagent B (µl)= [200 µl * ((number of samples x 3 replicates) + 5 standards] / 50
Reagent A (µl)= Reagent B *49
6. IMPORTANT: Keep samples and standards on ice. Prepare extra solution
7. Use a 96 well culture plate
8. Load 198 µl of the Reagent mixture into each of the wells used for sample, and 200
µl for standards
a. Use a small beaker and a Combitip with Repeater. Combitip can be reused
b. IMPORTANT: Rinse beaker and combitip before use
9. Load 2 µl of each sample/standard into the wells already containing reagent
mixture, and additionally 2 µl of RIPA to each standard (see Excel spreadsheet)
10. IMPORTANT: Tap gently the 96-well plate to mix sample and reagent mixture
11. Incubate at 37°C for 30 minutes (gently shaking)
Reading the proteins
1. Turn on the Plate reader, load the () file by pressing the icon in the top left.
2. Press Open/Close and place culture plate in the machine without its cover
(Plates are read from the top left corner to bottom right)
3. Press read
4. Verify that it is set to landscape before proceeding and that plate is in proper
orientation (record location in notes).
5. Save the results in the Franco Lab file.
6. Take a picture of the results screen and input to the Excel file () to calculate protein.
7. IMPORTANT: Do not forget to turn off the plate reader
3
Preparing samples and Running Gel
1. Determine protein concentration and mix them with the appropriate loading buffer.
Note that the sample volume determines the volume to be used for the rest of the
reagents
a. Laemmli buffer (LB)
i. Take 3 vol of sample (40 ul when you lysed the cells with 50ul)
ii. Thaw an aliquot of LB (400 ul), and add 100 ul B-mercaptoethanol (BME)
(in the fume hood).
iii. Add 1 vol of LB+ BME (i.e. 13.33 ul when you take 40 ul of sample)
iv. Vortex and incubate samples at 95oC for 5 min
4X Laemmli buffer (LB) (25 ml)

10 ml Tris HCl 0.5 M (200mM final) pH 6.8.
o Stock 0.5 M pH 6.8
 10 ml Glycerol (40 % final)
 2 g SDS (8 % final)
 8/100X25 =
 0.1 g Bromophenol blue (0.4 % final)
 NOTE: Dissolve and make up to 20 ml and aliquot into 50 tubes
(400 µl in 1.5 ml tubes).
 Thaw aliquots as needed and add 100ul ß- mercaptoethanol (20%
final) this will correct the missing volume.
b. 4x NuPAGE Loading/Sample buffer
i. Take 3 vol of sample (i.e. 40 ul)
ii. Add 1 vol of NUPAGE loading buffer 4X
iii. Add DTT from 1mM stock 20x
iv. IMPORTANT: It is better if you first prepare a “Master Mix” of NUPAGE
and DTT ([NUPAGE + DTT] x [# samples + 1 or 2 extra]) and then take
what is needed and add it to samples, this will decrease the pipetting
error
v. Vortex and incubate samples at 70oC for 10 min
NuPAGE Loading/sample buffer recipe
NuPAGE LDS Sample Buffer
106 mM
Tris HCl
141 mM
Tris base
2%
Lithium Dodecyl Sulfate
10%
Glycerol
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10ml 4x solution
0.666g
0.682g
0.800g
4.000g
4
0.51 mM EDTA
0.22 mM SERVA Blue G250
0.175 mM Phenol Red
pH = 8.5
Can be stored for 6 months at 4C
Buffer can be reused 3-5 times
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0.006g
0.750ml
(of 1% soln)
0.250ml
(of 1% soln)
ddH2O volume to 10ml
2. IMPORTANT: Place samples immediately on ice after being denatured
3. Centrifuge samples at 10,000 rpm (min^-1) 4°C for 5 minutes
4. Cast the gel system. See additional table. When pouring the gel remember to mix first all
components except for the TEMED. After addition of TEMED, mix well, pour into the
cast system and add a layer of isopropanol or H2O. Do not forget to check for leaks prior
to casting the gel.
5. IMPORTANT: Stacking gel has to be of at least 1cm high from the bottom of the well.
6. Wash wells thoroughly with running buffer
7. Fill chambers between the gels with running buffer. Fill exterior chamber about half
way with running buffer.
8. Load 2.5-5 µL of protein marker in first well.
9. Load the sample/loading buffer mix in the remaining wells according to the protein
determination (Use excel sheet)
10. IMPORTANT: Prepare a RIPA + loading buffer mix to fill empty wells, this will make the
gel run more even
11. IMPORTANT: Once the gel system is assembled do not move gels or chamber for any
reason as this might make the samples spill out of the wells
12. Make sure you connect red and black poles properly.
13. Start the electrophoresis at 80 volts (Amps max)
14. Once the samples are “in” the stacking gel and they are migrating in an even , well
defined, and compact band, you can increase to 100 and then to 200 volts.
15. IMPORTANT: Always monitor protein migration pattern, if bands start migrating unevenly decrease volts…..
10X Running buffer (Tris glycine) SDS PAGE (Biorad)
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30.28 G Tris base 250mM (25mM final)
142.61 g Glycine 1.90 M (190 mM final)
10g SDS 1% (0.1% final)
pH 8.3
Make up to 1L
NUPAGE MOPS and MES recipes
5
NuPAGE MOPS
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50 mM
MOPS
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50 mM
Tris base |
0.1%
SDS
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1 mM
EDTA
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pH = 7.7
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Good for 6 months at 4C
500ml 20x solution
104.6g
60.6g
10g
3g
ddH2O to 500ml
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NuPAGE MES
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50 mM
MES
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50 mM
Tris base |
0.1%
SDS
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1 mM
EDTA
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pH = 7.3
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Good for 6 months at 4C
500ml 20x solution
97.6g
60.6g
10g
3g
ddH2O to 500ml
6
Transferring the Gel to a Membrane
1. Cut membrane and filter paper to the same size as the gel. IMPORTANT: Avoid large
overhangs.
2. Soak the sponges and filter papers in the “Transfer Buffer”
3. If using PVDF, treat the membrane(s) with methanol for 1-2 min. Recycle methanol. You
do not need methanol in the transfer buffer for pvdf membranes
4. Remove the gel(s) from the glass plates and remove the small well plate section of the
gel. Cut out the stacking gel
5. Incubate the gel(s) and the membrane(s) in the “Transfer Buffer” for 10 minutes in ice
cold transfer buffer
6. IMPORTANT: Assemble gels in a submerged chamber. Get rid of the bubbles using the
roller
Towbin transfer buffer (1L).
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3.0 g Tris Base (25mM final)
14.4 g Glycine (192 mM final)
**1.0g SDS 0.1% (3.5 mM). Only for proteins larger than 80 kd. Reduce methanol
to 10% if SDS is required and add H2O to compensate for methanol. When
required, add 0.1 g SDS to 100 ml buffer.
Dissolve in 800 ml and store
Add methanol (20% of volume as required prior to use)
NuPAGE transfer buffer
25 mM
Bicine
25 mM
Bis-Tris
1 mM
EDTA
pH = 7.2
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125ml 20x solution
10.2g
13.1g
0.75g
ddH2O to 125ml
High Molecular Weight Proteins
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Add 0.05% SDS
Reduce methanol to 10%
Wet transfer ?
Semi dry transfer ?
7. Wet transfer (NUPAGE) (for wet transfer, it is important that the membrane is closest to
the positive electrode and the gel closest to the negative electrode):
7
a. For one membrane:
i. (top) Two Blotting Pads
ii. Filter Paper
iii. Membrane (the writing should be upside down and backwards in the
upper right hand corner)
iv. Gel
v. Filter Paper
vi. (bottom) two Blotting Pads
b. For two membranes:
i. (top) Two Blotting Pads
ii. Filter Paper
iii. Second Membrane
iv. Second Gel
v. Filter Paper
vi. Blotting Pad
vii. Filter Paper
viii. First Membrane
ix. First Gel
x. Filter Paper
xi. (bottom) Two Blotting Pads
c. Dump out the transfer buffer that has gotten into the outside container
d. Fill the inner chamber with transfer buffer
e. Put 500 mL of distilled water in the outside container
f. Run in the glass door refrigerator for 1 hour at 30 volts
8. Semi-dry transfer
8
a. Assemble gels as indicated below
b. Pour some transfer buffer on top of the gels
c. Determine area of transfer membrane and use the chart below to determine
current settings. Add extra 20 mA. Use the next formula: ([Area of membrane *
0.8 mAmps].
d. Transfer time is usually 1 h.
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9. After transfer, mark off the membrane(s) in the upper left hand corner (e.g. “LC3”, “1”,
“Franco”,etc.), and mark Protein Markers to avoid wash off their signal
10. After transfer, discard the gels, wash pads and/or filter papers in water
11. Wash 3 x 5-15 min each with TBS.
12. If required stain with Ponceau red solution for 1-2 min. Recycle Ponceau Red and wash
with destilled water to visualize protein bands.
13. Wash the remaining Ponceau stain with TBS
Ponceau S solution
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0.1% (w/v) Ponceau S (0.1 g in 100 ml)
5% (v/v) acetic acid (5 ml of acetic acid in 100 ml total)
Make up to 100 ml
Tris Buffer Saline (TBS) 10X
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87.7 g NaCl
12.114 Tris Base
pH 7.5 adjusted with HCl (pure)
Make up to 1L
10
TBS-T
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Dilute 10X TBS to 1X
Add 0.1% Tween-20 (1 ml per 1L)
14. Block the membrane at 4 oC overnight with 5 mL of blocking solution
a. Blocking solution: 5% milk (0.25 g) and/or 1-5% BSA in 5 ml TBS-T per membrane.
The blocking condition is stipulated by the antibody datasheet and bibliography.
IMPORTANT: BSA, but not milk, is recommended for phosho-proteins. Use a
plastic bag. Remember to get rid of bubbles
11
Incubation with Antibodies
1. Wash 3 x 5-15 min each with TBS-T ( a more thorough wash will clean up the
background better)
2. Add primary antibody in
a. Primary antibody buffer is usually the same as the blocking.
b. Use 3-5 mL of a 5% milk (0.25 g) and/or 1-5% BSA TBS-T mixture per membrane.
Again, the solution is stipulated by the antibody datasheet and bibliography and
usually matches the blocking buffer.
c. Antibody dilution is also stipulated in the corresponding datasheet (usually
1:1000. Actin is 1:5000)
d. IMPORTANT: For phosho-proteins, BSA, but not milk is recommended.
e. Primary antibodies are re-used at least 3-times and stored at -20oC.
f. Use a plastic bag. Remember to get rid of bubbles and reseal the bag
3. Time of incubation can go from 1-2 h to overnight at 4 oC. Actin and GADPH (loading
controls) are usually incubated for a couple of hours max.
4. Wash 3 x 5-15 min each with TBS-T (a more thorough wash will clean the background
better)
5. Add secondary antibody in 3-5 mL of a 5% milk (0.25 g) and/or 1-3% BSA TBST mixture
per membrane. Use a plastic bag. Remember to get rid of bubbles and seal the bag.
6. IMPORTANT: If the primary antibody’s host is rabbit, use anti-rabbit for secondary, if the
primary antibody’s host is mouse, use anti-mouse for secondary.
7. Secondary antibody dilution goes from 1:5000 to 1: 8000. Secondaries are re-used for a
whole week and kept at 4oC.
8. Incubate secondary antibody at RT for 2 h.
9. Wash 3 x 5-15 min each with TBS-T.
12
Developing the Membrane
1. Use Pierce ECL Western Blotting Substrate.
2. IMPORTANT: There is an ADVACE/PLUS/ECL 2 substrate which is more sensitive, do not
use this one unless required. See below
3. Mix 1:1 vol of Reagent A and B. For 1 membrane use 500ul total (250ul of A and 250ul of
B). For more than 1 membrane use 1 ml total (500ul of A and 500ul of B). Do not mix
tips or stock solutions. Always keep stocks closed
4. Wash membrane with the ECL substrate using a 1000ul micropipette.
a. When using the developing machine:
i. Put the membrane on a sheet protector within an exposure cassette
ii. Dry off the excess substrate
iii. Take it to the dark room (The machine must be turned on 20 minutes in
advance. It is best to do this while the membrane is being washed).
Follow instructions located right next to the developing machine
iv. IMPORTANT: Lock the room to avoid somebody opening the door. Always
remember to handle films in the dark under the red light
v. Expose one film for 2 minutes and develop and adjust subsequent
exposures as needed
vi. IMPORTANT: Do not forget to label protein markers to identify targeted
protein
vii. When done, turn-off machine and wash membrane with TBS-T
b. When using the Versa:
i. Follow instructions right behind the machine.
ii. Use Quantity One software and select Versa as Detector
iii. Use the positioned to locate membrane with the door open
iv. Close the door and do single expositions preferably
v. Clean the machine when done. Wash membrane with TBS-T
vi. Do not turn off the equipment.
5. When using ADVANCE/PLUS/ECL 2 substrate, start with a 1:5 or 1: 10 dilution of a 1:1
mixof reagent A and B.
a. For example. For a 1:5 dilution
i. Prepare 500 ul of substrate by adding 400 ul H2O + 50ul Reagent A + 50
ul Reagent B
b. For a 1: 10 dilution
i. Prepare 500 ul of substrate by adding 450 ul H2O + 25ul Reagent A + 25
ul Reagent B
c. If required wash once with TBS-T to reduce background
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14
Membrane Stripping
1. Incubate membrane with Stripping buffer for 45 min at 50oC
2. Wash 3 x 5-15 min each with TBS ( a more thorough wash will clean the background
better)
3. Proceed to blocking
Harsh stripping solution (500 ml)
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100 ml SDS 10% (1% final)
62.5 ml Tris HCl pH 6.8 0.5M (62.5 mM final)
67.5 ml ultra pure water
Make up to 500 ml
Prior to use add 80ul β-mercaptoethanol per 10ml solution
Re-use buffer at least 3 times