SOP Protein Extraction and Western Blotting Starr Lab
Title or Type of Procedure: Extraction of protein from human and mouse cells or
tissues and Western Blotting
P. I. Timothy K. Starr
Lab Location: 12-137 MoosT
Original Issue Date: 4/12/11
Prepared By:
Revision Date:
5/21/11, 3/4/14
Timothy K. Starr Approval Signature:
(if required by lab supervisor)
Procedural Methods and Materials:
General Safety Procedures:
Wear Personal Protective Equipment (PPE): sterile gloves, eye goggles and lab coat.
Work involving beta-mercaptoethanol should be performed in a Fume Hood
Materials and Procedure
Cell lysate protein prep
Wash adherent cells twice in the dish or flask with ice-cold PBS and drain off PBS. Wash
non-adherent cells in ice cold PBS and centrifuge at 800 to 1000 rpm in a tabletop centrifuge for 5 minutes to pellet the cells.
Add ice-cold modified RIPA buffer to cells (1 ml per 107 cells/100 mm dish/150 cm2
flask; 0.5 ml per 5 x 106 cells/60 mm dish/75 cm2 flask).
Scrape adherent cells off the dish or flask with either a rubber policeman or a plastic cell
scraper that has been cooled in ice-cold distilled water. Transfer the cell
suspension into a centrifuge tube. Gently rock the suspension on either a rocker or
an orbital shaker in the cold room for 15 minutes to lyse cells.
Centrifuge the lysate at 14,000 x g in a precooled centrifuge for 15 minutes. Immediately
transfer the supernatant to a fresh centrifuge tube and discard the pellet.
Supernatant contains cytoplasmic proteins while pellet contains nuclei & debris.
Nuclear proteins are still present, although at a reduced amount.
Dilute the cell lysate at least 1 : 10 before determining the protein concentration because
of the interference of the detergents in the lysis buffer with the Coomassie-based
reagent. At this step, the sample can be divided into aliquots and stored at -20ºC
for up to a month.
TIP: When working with large volumes of non-adherent cells, the cells may not be
cooled quickly enough to maintain the activity of the protein being studied. In this
case, pour the cell suspension into a mixture of an equal mass of 2 x PBS and ice,
then collect the cells by centrifugation and perform the lysis as described above.
Lysis Buffer recipe (a.k.a. RIPA buffer - radioimmunoprecipitationassay). Normally
used for post-nuclear lysate preps.
1x – 1 ml
Final conc
50 µl
1 M Tris pH 7.5
[50 mM]
30 µl
10 µl
5 M NaCl
Nonident P40
[1 %]
[150 mM]
Variation: Triton X-100
2 µl
500 mM EDTA pH 7.4
[1 mM]
Variation: pH 8.0
10 µl
10% SDS
[0.1 %]
5 µl
200 mM EGTA
[1 mM]
Optional
50 µl
10% Na-deoxycholate [0.5%]
Optional: a.k.a deoxycholate acid
sodium salt
subtotal volume = 157 µl
778 µl H2O
Prepare the above stock and then add the following inhibitors just before using.
40 µl
Protease Cocktail
[?]
1 pill in 2ml H2O
10 µl
100 mM PMSF
[1 mM]
see note below
10 µl
1 M NaF
[10 mM]
5 µl
200 mM Na3VO4
[1 mM]
See Activation note below
subtotal volume = 65 µl
Note: Can substitute the following protease inhibitors for the cocktail pill above
1 µl
10 mg/ml aprotinin
[1 µg/ml]
See Variation below:
1 µl
10 mg/ml leupeptin
[1 µg/ml]
1 µl
10 mg/ml pepstatin
[1 µg/ml]
Variation: 2 µg/ml
Variation: Can use a Complete Protease Inhibitor Cocktail tablet in place of the
aprotinin, leupeptin and pepstatin. Currently using Roche Diagnostics tablets (Cat
# 1 697 498). One tablet is dissoved in 2 ml H2O and stored at –20º C. Use 40
µl/ ml of buffer. These tablets require EDTA at 1 mM in the buffer.
Na3VO4 is added to inhibit removal of phosphate groups. The activity of Na3VO4 can
be substantially increased by the following activation procedure:
Make 200 mM stock (0.368 g/ 10 ml H2O)
Adjust to pH 10 using HCl or NaOH
Boil until colorless (approx 10 min)
PH to 10 again
Repeat boiling and pH adjustment until liquid is colorless and pH stabilizes
Aliquot and store at –20º C.
PMSF is extremely unstable (30 min) in aqueous form. So stock is made up in
isopropanol and stored at –20º C and then added just before using. To make 10
ml of 100 mM add 0.174 g PMSF to 10 ml isopropanol.
Store samples at – 80º C. Boil for 10 minutes before loading onto gels.
Western blotting
Preparing the gel
You can either buy the Invitrogen NuPage prepoured gels, or pour your own gel using
Invitrogen Gel Cassettes (1.0mm cat# NC2010). Or use any other electrophoresis
aparatus. The gel we currently use is a Bis-Tris acrylamide gel. Generally I use a
10% acrylamide gel, but you can vary the percentage depending on the size of the
protein you are looking for.
Separating Gel Recipe to make 4 gels (1.0 mm thick), approx 25 ml. (Adapted from
invitrogen).
10% gel
6.25 ml
9.4 ml
250 µl
4 ml
edge
4.5 ml
final conc
40% Acrylamide/Bis-Acryl 29:1
1 M Tris pH 8.8
10% SDS
50% sucrose
[10%]
[375 mM]
[0.1 %]
[8 %]
optioinal: better gel
H2O
8% gel = 5 ml acryl/bis, 5.8 ml H2O
12% gel = 7.5 ml acryl/bis, 3.3 ml H2O
6.25 µl TEMED
625 µl
5% APS
APS
[0.025 %]
Variation: 5 µl TEMED
[0.125 %]
Variation: 125 µl 10%
Add the TEMED and APS last, as they will start the solidification of the gel.
Make APS (ammonium persulfate) fresh each time. To make 5% APS add 100 mg to 2
ml H2O
Using a 10 ml pipette, fill gel casting 3/4 full (~ 6 ml).
Using a 1 ml pipette, overlay with H2O, filling casette to top very gently. Variation:
overlay with butanol.
Let gel solidify for about 30 – 60 min. Pour off H2O and dry w/Kim wipe. Variation,
pour off butanol, rinse with H2O and dry.
Stacking Gel Recipe to make 4 gels (1.0 mm thick), approx 12.5 ml
4% gel
1.25 ml
4.2 ml
125 µl
6 ml
final conc
40% Acrylamide/Bis-Acryl 29:1
[4 %]
375 mM Tris pH 6.8
[126 mM]
10% SDS
[0.1 %]
H2O
5.0 µl
1.0 ml
TEMED
5% APS
[0.04 %]
[0.4 %]
Add the TEMED and APS last, as they will start the solidification of the gel.
Using a 10 ml pipette, fill gel casting to the top (~ 3 ml).
Gently insert comb into gel casting.
Let gel solidify for about 30 – 60 min.
All buffers can be stored indefinitely at RT, except Acryl/Bis, TEMED & APS.
Gels can be stored at 4ºC for several weeks if wrapped in cellophane with a wet paper
towel.
WB: Prepare electrophoresis chamber
Prepare running buffer. Can use several different types of buffers depending on
the gel you are using. Read the Invitrogen “Using the XCell II Blot
Module” manual for more information.
Assemble the electrophoresis chamber with one or two gels. Be sure to remove
the tape strip at the bottom of the gels.
For NuPage prepoured gels, or our own BIS-Tris prepoured gel use MOPS
based buffer purchased from invitrogen (NP0001) 40 ml 20x buffer into
760 ml H2O. Otherwise use following running buffer .
Running Buffer (SDS-PAGE) recipe
1x
10x
3g
30.2 g
Tris
14.4 g
144 g
Glycine
2g
20 g
SDS
up to 1 L
up to 1 L
H2O
1x conc
[25 mM]
[192 mM]
[0.2 %]
Variation: 1 g [0.1 %]
Fill inner chamber first and check to make sure there are no leaks. Then fill outer
chamber (fill full if using NuPage buffer, otherwise can just cover the
bottom holes). Add 500 µl of NuPage anti-oxidant to inner chamber if
using NuPage buffer.
Remove comb and clean out wells by pipetting up and down to get rid of chunks
of gel that
WB: Load Protein sample:
Prepare protein lysates following protein lysate protocol. Generally need about
20 – 50 µg of protein per lane. If using a 10 lane gel, the lane will hold
about 40 µl, which means you can use 32 µl protein lysate + 8 µl of 5x
sample running buffer. Or, if you have prepared your sample using
sample running buffer, you can load the lysate directly.
Combine lysate, H2O and 8 µl 5x Laemmli buffer to total volume of 40 µl. Pulse in the
centrifuge. Boil for 10 minutes. Pulse in the centrifuge.
One lane should include a purchased prestained protein ladder. Currently using
Benchmark Prestained Protein Ladder, but many others are available. Remember
they are not always accurate when used on different gel types or percentages.
These ladders need to be boiled, but already contain Laemmli buffer.
Load sample into wells using gel loading pipette tips. Normally load about 20 to 50 µg
of protein, or 3 to10 e6 cell equivalents.
5x Laemmli buffer recipe (Comes from Maniatis A8.42 Cell lysis 1xSDS buffer)
10 ml
1 ml
100 ml
5x conc
1x conc
1g
0.1 g
10g
SDS
[10%]
[1 %]
5 ml
500 µl
50 ml
glycerol
[50%]
[10%]
3.0 ml
300 µl
30 ml
1 M Tris pH 6.8
[300 mM]
[60
mM]
0.05 g
0.005g 0.5 g
Bromophenol [0.005%]
[0.001 %]
2 ml
200 µl
20 ml
H2O
Can make up 10 ml and aliquot in 500 µl, store in frig.
Just before using add (DTT stored in soluble form loses its reducing potential)
0.76 g
0.076 g
7.6 g
DTT
[0.5 M]
Alternatively, just before using add 7 to 25 µl beta-ME [1.4 - 25%] to 500 µl.
Run gel: times and voltages depend on gel type. For NuPage prepoured gels and MOPS
running buffer can run at 200 V for about 50 minutes (see Invitrogen manual).
Otherwise run at 100 – 125 V for 1 to 2 hrs. Can start with 80 V for 10 minutes to
get even entry into the gel. Stop when dye front reaches bottom of gel.
WB: Prepare transfer apparatus
Three types of membranes are available for transferring: Nitrocellulose, PVDF, and
nylon. Generally, nitrocellulose is easiest to use while PVDF has better
properties. Membranes can have different pore sizes and some membranes have
front and back sides. Currently I am using PVDF membranes. Immun-Blot
PDVF membrane 0.2µm from BioRad.
Keep track of which side is up. PVDF-plus membranes have a shiny side and a dull side
because the pores are actually cone-shaped. Small end of cone is shiny side,
while large end is dull. On the BioRad roll, the dull shiny side is on outside of
roll, dull side is on inside. I try to put the dull side (large end of pores) facing the
gel. When I do washes and everything else, I keep this side facing up. According
to mfcturer it doesn’t matter as long as you are consistent.
Cut membrane to be same size as gel. Activate the PVDF membrane. Place in a tray
with methanol for 15 seconds, transfer to a tray with water for 2 minutes, and
finally place in a tray with transfer buffer for 5 minutes.
Soak blotting pads and 3 mm Whatman filter paper (usually 4 pieces of paper per gel) in
transfer buffer.
When gel has finished running, crack open the gel cassette using the pry knife. I try to
pry off the smaller side of the gel cassette so the gel is resting on the larger
cassette. Cut off the stacking gel portion of the gel. Pipette about 1 ml of transfer
buffer onto the gel. Place two Whatman filter papers on top of the gel, making
sure there are no bubbles. Flip the whatman filter paper/gel/cassette over onto
parafilm or clean desktop. Using the pry knife separate the cassette from the
gel/filters. It may be necessary to push the gel foot out of the cassette. Be gentle.
Once you have removed the cassette, pipette another 1 ml of transfer buffer onto
the gel.
Place the PVDF membrane (dull side facing gel) onto the gel. You may want to cut a
corner of the membrane before placing it on the gel for easy identification later.
Make sure there are no bubbles. Pipette another ml of transfer buffer onto the gel
and place two more Whatman filter papers on top of the gel.
Put two soaked blotting pads onto the Anode side of the blot module. (This is the boxlike side of the module). Place the filter/gel/membrane/filter sandwich on top of
the two blotting pads. Remember: Gel is towards the anode (-), membrane is
towards the cathode (+), because proteins are coated with negatively charged SDS
and will migrate towards the cathode. Put on more blotting pad and then another
filter/gel/membrane/filter sandwich if you have a 2nd gel. Put enough remaining
blotting pads to fill up the anode box. Place the cathode top on the box, squeeze
together and insert into the box. Fasten clamp.
Pour enough transfer buffer into chamber so that blotting pads are covered + 0.5 cm
above (don’t go all the way to the top). Fill the outer chamber with water.
Run transfer at 30 V for 1 hr. Note, different voltages and times are required for different
gels and membranes. You can check to see whether or not transfer has occurred
by opening blot module, gently lifting corner of the membrane to check whether
or not the protein ladder proteins have transferred.
Transfer buffer recipe depends on type of gel you are transferring from and on the
membrane. The following recipe is for 1 liter for PVDF membrane & Bis-Tris
gels:
Transfer Buffer Recipe
1x
10x
3g
30.2 g
14.4 g
144 g
100 ml (add after dilution)
200 ml (add after dilution)
0.5 g
5g
proteins
92.5 mg
phosphotyrosine
up to 1 L
up to 1 L
1x concentration
TRIS
[25 mM]
Glycine
[192 mM]
Methanol
[10%]
For transferring 1 gel
Methanol
[20%]
For transferring 2 gels
SDS
[0.05%]
Optional for big
Na3VO4
[0.5 mM]
Optional for
H2O
Transfer buffer solution should be degassed (i.e. let it sit overnight).
Alternatively, Invitrogen’s NuPage 20x transfer buffer is as follows:
20x
10.2 g
13.1 g
0.75 g
0.025 g
up to 100 ml
1x concentration
Bicine
[25 mM]
Bis-Tris (free base)
[25 mM]
EDTA
[1 mM]
Chlorobutanol [0.05 mM] Optional preservative for long storage
H2O.
To make NuPage transfer buffer from 20x stock prepared above:
1 liter
600 ml
50 ml
30 ml
20x NuPage
1 ml
600 µl
NuPage antioxidant
100 ml 60 ml
Methanol
[10 %]
For transferring 1 gel
849 ml 509 ml H2O
200 ml 120 ml Methanol
[20 %]
For transferring 2 gels
749 ml 449 ml H2O
WB: Block membrane
Place membrane in container (plastic weigh boat, pipette tip box, seal-a-meal bag) and
rinse one or two times with TBS-T (or TBS), then add enough blocking buffer to
cover. Rock for 1 hr RT.
TBS Buffer pH 7.4
8g
0.2 g
3.0 g
0.015 g
up to 1 L
NaCl
KCl
TRIS
Phenol red
H2O
[137 mM]
[2.68 mM]
[25 mM]
[40 nM]
variation: 50 mM
optional
TBS-T = Add Tween20 to desired concentration, normally 0.5%, but can be 0.01 to
0.1%.
Blocking Buffer = TBS-T plus some random protein. Often use 2 to 5% milk powder or
Bovine Serum Albumin. Can also add goat IgG or other blocker if want to.
WB: 1º Antibody staining
Pour off blocking buffer and replace with blocking buffer + 1º Ab. Concentrations
of primary Ab run from 1:100 up to 1:10,000. Try to find someone who
has already done this and use that as the starting concentration. Place on
rocker for 1 to 12 hours at RT or at 4º C if going for a long time.
Wash with TBS-T. This should be done at least three times over a period of at
least 1 hour, but can be longer with more rinses in between. Can do initial
rinses in TBS.
WB: 2º Antibody staining
Replace wash with 2º antibody in blocking buffer at appropriate concentration
(generally from 1:1000 to 1:10,000 or 1:100,000 for monoclonal 2º’s). 2º
antibody is normally conjugated either to Horseradish Perioxidase or to
Alkaline Phosphatase. These enzymes will then cleave a substrate that
can be detected either on film or using a scanner.
Wash with TBS-T, similar to 1º antibody staining.
WB: Chemilumescence detection
Prepare chemiluminscent substrate. Mix equal parts of bottle A and bottle B from Super
Signal West PICO chemiluminescent substrate kit. Need about 10 ml for one gel.
Incubate membrane in 10 ml of the chemiluminscent substrate for 5 min. in fresh
weigh boat on rocker.
Prepare film apparatus. Put saran-wrap covered whatman paper on bottom of apparatus.
Place the glowing stickers on the saran wrap on the top and side of where the membrane
will be placed. Place the membrane with the side that was facing the gel facing
up. Place another piece of saran wrap on top of the membrane.
Take the picture. In the dark room, turn off lights, place a sheet of film over the
membrane (try to do this smoothly, without moving the film or membrane) and
cover for appropriate length of time (1 second to 1 minute to longer). Put film in
the developing machine. Note: The chemiluminescent stuff will gradually get
brighter for about a half hour and then gradually get darker, so the developing
time is really an artistic guess. Wait to see if picture develops. Take another if it
didn’t come out well.
Hazard Identification and Risk of Exposure to the Hazards:
The two main hazards for this protocol are exposure to skin or mucous membranes by
material from tissue or cells or exposure to toxic reagents.
Exposure Controls Specific to Above Risk of Exposure:
PPE - Lab coats, safety goggles, mask, gloves, sleeves and sharps containers.
If exposed to chemicals or tissues/cells wash with water and detergent and seek medical
attention if warranted. Bring MSDS to area where seeking medical attention. If you
suspect that a natural barrier may be broken and internalization of tissue components may
have occurred, seek medical attention. Although the use of sharps are not specifically
called for in this protocol, if one uses them in the homogenization step, do not recap
needles and dispose of all sharps in designated sharp containers.
Waste Generated and Disposal Methods:
Liquid waste in contact with cells or tissues will be collected in a flask or beaker
containing bleach 10% (v/v) and will soak for 30 minutes before being sewered.
Liquid waste from buffers of Western blot (transfer buffer, blocking buffer, antibody
staining buffer) are safe to dispose of in sink.
Solid waste in contact with cells or tissues should be discarded in a biohazard bag for
incineration. Other solid waste can be discarded in the trash.
Sharps containers will be sealed when ¾ full and placed in designated waste area.
Refer to the Biological Waste Disposal procedures posted on the Tissue Culture room
door for more information
Spill Response Procedures:
Cover any spills with paper towels, and place towels in bucket labeled as hazardous
waste. If exposed to chemicals or tissues/cells immediately rinse with copious amounts
of water and seek medical attention if warranted at Boynton Health Services, or Fairview
Emergency room if after normal business hours.
Accident Response Procedures:
If Incident Results in a Hazard Exposure ( i.e. face or eye splash, cut or puncture with
sharps, contact with non-intact skin):
• Encourage needle sticks and cuts to bleed, gently wash with soap and water for 5
minutes; flush splashes to the nose, mouth, or skin with water; and flush eyes at the
nearest eyewash station with clean water for 15 minutes.
• Call 911 or seek medical attention.
- For urgent care employees may go to HealthPartners Occupational and
Environmental Medicine (M/F day time or Urgent Care after hours), or UMMCFairview Hospital (24 hrs). You may seek medical attention at the closest available
medical facility or your own healthcare provider.
- Follow-up must be done by HealthPartners Occupational and Environmental
Medicine.
• Report the incident to your supervisor as soon as possible, fill out the appropriate
documentation.
- Employee First Report of Injury
- Supervisor Incident Investigation Report
• Send Incident Report Form to the IBC if exposure has occurred during work on an
IBC protocol.
• Report all biohazard exposures to the Office of Occupational Health and Safety (612626-5008) or uohs@umn.edu.
Note: It is important to fill out all of the appropriate documents to be eligible to collect
workers compensation should any complications from the hazardous exposure arise in
the future.
Notes: (special record keeping such as inventories for toxins, reporting, training, etc.
that may be required)
References:
For further information view the UMN DEHS website containing Bio Basic Fact Sheets
at http://www.dehs.umn.edu/bio_basicfacts.htm.
For general information on Biosafety, access the Biosafety in Microbiological and
Biomedical Laboratories (BMBL) 5th Edition from the CDC at BMBL
http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
For Material Safety Data Sheets access the Public Health agency of Canada website
MSDS http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php.