Simple Western Compatibility Table
with Commercial Lysis Buffers
Highest
Dilution Tested
Assay signal
Effect on
Fluorescent Standards
Resolution
Lysis buffer
Vendor
Recommendations
Components
RIPA
Cell Signaling
(p/n 9806S)
1X (100%)
Possible signal decrease in
proteins >150kDa, compared to
samples in Simon Sample
Buffer, other proteins not
affected
Non-linear compression in low
No Effect
MW region, causing the 1 and 12
kDa fluorescent standards to run
closer to each other.
Signal decrease in 180kDa
fluorescent standard.
Samples can be used undiluted with Simon 10X
Sample Buffer/Fluorescent Standards mix*.
Use ladder in the same buffer for consistent sizing.
For best signal and resolution dilute 1:2 in Simon
Sample Buffer
Cell Lysis Buffer
Cell Signaling
(p/n 9803S)
1X (100%)
Possible signal decrease in
proteins >150kDa, compared to
samples in Simon Sample
Buffer, other proteins not
affected
Non-linear compression in low
No Effect
MW region, causing the 1 and 12
kDa fluorescent standards to run
closer to each other.
Signal decrease in 180kDa
fluorescent standard.
Samples can be used undiluted with Simon 10X
Sample Buffer/Fluorescent Standards mix*.
Use ladder in the same buffer for consistent sizing.
For best signal and resolution dilute 1:2 in Simon
Sample Buffer
20mM TrisCl pH 7.5,
150mM NaCl,
1mM EDTA,
1mM EGTA,
1% NP40,
1% Sodium deoxycholate,
Protease/phosphatase inhibitors cocktail
20mM TrisCl pH 7.5,
150mM NaCl,
1mM EDTA,
1mM EGTA,
1% Triton,
Protease/phosphatase inhibitors cocktail
SDS Lysis Buffer
Millipore
(p/n 20-163)
0.7X (70%)
No Effect
Significant signal decrease for
1kDa Fluorescent Standard and
registration point
IP Lysis buffer
Pierce
(p/n 87787)
0.7X (70%)
T-Per
Pierce
(p/n 78510)
0.7X (70%)
Possible signal decrease in
proteins >150kDa, compared to
samples in Simon Sample
Buffer, other proteins not
affected
No Effect
Non-linear compression in low
No Effect
MW region, causing 1 and 12 kDa
standards to run close to each
other. Signal decrease in high
MW standards.
No effect, compared to Simon
No Effect
Sample Buffer
70% samples with 10X Simon Sample Buffer /FLS/DTT
have good signal, but may have problems with
registration**.
For best sizing and resolution dilute 1:2 in Simon
Sample Buffer. Use ladder in the same buffer for
consistent sizing.
Samples can be used undiluted with Simon 10X
Sample Buffer/Fluorescent Standards mix*.
Use ladder in the same buffer for correct sizing.
For best signal and resolution dilute 1:2 in Simon
Sample Buffer
Samples can be used undiluted with Simon 10X
Sample Buffer/Fluorescent Standards mix.
Cellytic MT
Sigma
( p/n C3228)
0.7X (70%)
No Effect
0.7X (70%)
N/A
Mild Non-linear compression in No Effect
low MW region, causing 1 and 12
kDa standards to run closer to
each other.
Signal decrease for all
N/A
Fluorescent Standards, especially
for 1kDa
Commercial
Cell Signaling
lysates prepared in (various lysis
various lysis
buffer)
buffers
No Effect
Samples can be used undiluted with Simon 10X
Sample Buffer/Fluorescent Standards mix.
Samples may have problems with fluorescent
standards assignment and registration, manual
assignment may be necessary.
For best sizing and resolution dilute 1:2 in Simon
Sample Buffer.
Use ladder in the same buffer for consistent sizing.
All results are compared to performance of standard Simon Sample buffer (20mM Bicine pH 7.6/0.6% CHAPS/1%SDS/40mM DTT)
* MW sizing in samples containing TritonX-100 and NP-40 is affected by non-linear compression of low MW Fluorescent standards.
This effect may be ameliorated by substituting 10X Simon Sample Buffer with SDS only, adding it to a final concentration of 1%.
** SDS at >1% may affect resolution and 1 kDa fluorescent standard signal. This effect may be ameliorated by substituting 10X Simon Sample Buffer CHAPS only, adding it to a final concentration 0.6%.
Rev 1 November 2011
50mM TrisCl pH 8.1,
10mM EDTA,
1%SDS
25mM TrisCl pH 7.4,
150mM NaCl,
1mM EDTA,
1% NP40,
5% glycerol
25mM Bicine pH 7.6,
150mM NaCl,
Proprietary detergent
Bicine, unknown concentration,
150mM NaCl,
proprietary dialysable mild detergent
62.5mM TrisCl, pH 6.8,
2%SDS,
10% Glycerol
Simple Western Reagent Compatibility Table
Buffering Reagents
Component
Tris-Cl
HEPES
Concentration
Range Tested
Usage
Commonly used as buffering agent in
50-100mM at pH
commercial cell lysis buffers at 10-50mM, at
7.2
pH 6.8-8.0
Used as buffering agent in some cell lysis
buffer, and nuclear protein extraction
20-40mM at pH 7.5
buffers
Sodium phosphate Occasionally used as buffering agent in cell
(NaH2PO4/Na2HPO4)
lysis buffers at 10mM
Concentration Range
Recommended
Effect on Signal
Sizing/Resolution
10-100mM
No Effect
No Effect
20-40mM
No Effect
No Effect
20-40mM
No Effect
No Effect
Effect on Signal
Signal Decrease with
>300mM, especially for
high MW proteins.
Less effect on small
proteins.
Sizing/Resolution
10-20mM
Salts
Concentration
Range Tested
Concentration Range
Recommended
Component
Usage
Nacl
Commonly used to control osmolarity in
commercial cell lysis, and Laemmli buffers
at 100-150mM
100mM - 1M
0-200mM
NH4Cl
Used in RBC lysis buffer as osmolarity agent
at 150mM
150mM -300mM
0-300mM
Alignment problems due to
strong decrease of 180kDa
Fluorescent standard
No Effect
No Effect in tested range
Effect on Signal
Signal Increase for some
Small Proteins
Signal Increase for Some
Small Proteins
No Negative Effect,
Increase in signal for small
proteins
Sizing/Resolution
Non-linear compression occurs in
low MW region, causing 1 and 12
Non-linear compression occurs in
low MW region, causing 1 and 12
Detergents
Component
TritonX-100
NP40
C7BzO
Usage
Non-ionic detergent commonly used in
commercial cell lysis buffers at 0.5- 1%
Non-ionic detergent commonly used in
commercial cell lysis buffers at 0.5- 1%
Zwitterionic detergent commonly used in
commercial cell lysis buffers at 0.5- 1%
Zwitterionic detergent used in commercial
cell lysis buffers at 0.5- 1%
Ionic detergent, RIPA buffer component at
Sodium Deoxycholate
0.5-1%
Ionic detergent commonly used in
SDS
commercial cell lysis, and Laemmli buffers
at 1-2%
CHAPS
Concentration
Range Tested
Concentration Range
Recommended
1-2%
0-1%
0.5-1%
0-0.5%
1-2%
0-2%
0.6-2%
0.6-2%
No Effect
0.6-1.2%
0-0.6%
Signal Increase for some
small proteins
1-3%
1-2%
No Significant Effect
Slight decrease in resolution at
>1%, strong decrease in
resolution at>2%
No Effect in tested range
Exacerbates MW shift with high
protein concentration sample,
Reducing Agents
Component
Usage
Concentration
Range Tested
Concentration Range
Recommended
DTT
Common reducing agent , used at 5-50mM
40-80mM
Effect on Signal
Sizing/Resolution
40-80mM
No Effect
BME
Common reducing agent , used at 200mM
to 1M
No Effect
200-400mM
200-400mM
TCEP
Reducing agent , used at 0.5-10mM
1-20mM
1-20mM
Component
Usage
Concentration
Range Tested
Concentration Range
Recommended
Glycerol
Laemmli buffer component, at 10%
10-20%
0-20%
Sucrose
Used in cell lysis buffers for subcellular
fractionation at 250-300mM
250mM-500mM
0-500mM
Signal increase for some
small proteins
Signal increase for some
small proteins
No Effect
No Effect
Viscosity Reagents
Effect on Signal
No Negative effect,
Increase in signal for small
proteins
Sizing/Resolution
No Effect
No Effect
Effect on Signal
Sizing/Resolution
No Effect
Miscellaneous Reagents
Component
Usage
Concentration
Range Tested
Concentration Range
Recommended
Urea
Occasionally used to achieve higher protein
yield, at 7-9M
0.5M-8M
0-1M (varies for
different proteins,
some may be less
sensitive)
Urea/Thiourea
Occasionally used to achieve higher protein
yield, at 7M Urea/2MThiourea
2MUrea/0.5M
Thiourea 4MUrea/1M
Thiourea
0-2MUrea/0.5M
Thiourea (varies for
different proteins,
some may be less
sensitive)
MgCl2
EDTA
Rev 1, November 2011
Signal Decrease with >1-2M
urea (varies for different
proteins), large proteins
may benefit from addition
of 0.5M urea)
Signal Decrease with
>2MUrea/0.5M Thiourea
(varies for different
proteins), large proteins not
affected even at
4MUrea/1M Thiourea)
Used in nuclear protein extraction and
3-6mM
0-6mM
No Effect
immunoprecipitation buffers at 1-3mM
Used in commercial cell lysis buffers to
deactivate metal-dependent enzymes, at
2.5- 10mM
0-10mM
No Effect
0.5-2.5mM
All results are compared to performance of standard Simon Sample buffer
No Effect
No Effect
No Effect
No Effect
2
How buffer components were tested:
Buffers prepared as 1.4X solution; than sample, DTT, FL standard added at 10X
Initial test on CB1000 (MV205)
K562 denatured at 0.8 mg/ml, or Hela denatured at 1mg/ml
duplicates repeated in 2 cycles
Samples probed with 4EBP1, ERK1/2 and PLCg
When effect of the additive was observed, samples were retested, with extended dilution range
Composition of all the buffers tested:
Tris-HCl (50mM, 100mM)/0.6%CHAPS/1%SDS/40mMDTT
HEPES(20mM, 40mM) /0.6%CHAPS/1%SDS/40mMDTT
0.01 M sodium phosphate (NaH2PO4/Na2HPO4), pH 7.2/0.6%CHAPS/1%SDS/40mMDTT/0.15M NaCl
0.01 M sodium phosphate (NaH2PO4/Na2HPO4), pH 7.2/0.6%CHAPS/1%SDS/40mMDTT
20mM Bicine/0.6%CHAPS/1%SDS/40mMDTT/NaCl (100mM,250mM, 500mM, 1M)
20mM Bicine/0.6%CHAPS/1%SDS/40mMDTT/NH4Cl (150mM, 300mM)
20mM Bicine/0.6%CHAPS/1%SDS/40mMDTT/Glycerol(10%,20%)
20mM Bicine/0.6%CHAPS/1%SDS/40mMDTT/Sucrose (300, 600mM)
20mM Bicine/TritonX(1%,2%)/1%SDS/40mMDTT
20mMBicine/NP40(1%,2%)/1%SDS/40mMDTT
20mMBicine/Sodium deoxycholate (0.5%,1%)/1%SDS/40mMDTT
20mMBicine/C7BzO(0.5%,1%)/1%SDS/40mMDTT
Bicine/CHAPS(0.6%,1.2%)/1%SDS/40mMDTT/
20mMBicine//0.6%CHAPS/SDS(1%,2%,3%)/40mMDTT/
20mM Bicine/0.6%CHAPS/1%SDS/DTT(40mM, 80mM)
20mM Bicine/0.6%CHAPS/1%SDS/BME (2mM, 4mM)
20mM Bicine/0.6%CHAPS/1%SDS/40mMDTT/TCEP (1mM-20mM)
20mM Bicine/0.6%CHAPS/1%SDS/40mMDTT/MgCl2 (3mM,6mM)
20mM Bicine/0.6%CHAPS/1%SDS/40mMDTT/EDTA(5mM, 10mM)
20mM Bicine/0.6%CHAPS/1%SDS/40mMDTT/Urea/Thiourea (2M/0.5M; 4M/1M)
20mM Bicine/0.6%CHAPS/1%SDS/40mMDTT/Urea (0.5, 1, 2, 4, 8M)
Rev 1, November 2011
3