Amanda Clouser
2/13/16
HSP27 Full-Length Purification Protocol
DAY 1- Starting with 1L culture resuspended in 15 mL lysis buffer (50 mM Tris/HCl,
100 mM NaCl, 1 mM EDTA, pH 8.0) and stored at -80C
MATERIALS
 Prepare 3L of “HSP27 Buffer 1” (20 mM Tris/HCl, 10 mM MgCl2, 30 mM
NH4Cl, 0.5 mM EDTA, 0.5 mM DTT, pH 7.6)
o 9.46g Tris/HCl (157.6g/mol * 0.02mol/L * 3L)
o 6.10g MgCl2  6H2O (203.31g/mol * 0.01mol/L * 3L)
o 4.81g NH4Cl (53.49g/mol * 0.03mol/L * 3L)
o 43.84 EDTA (292.24g/mol * 0.050mol/L * 3L)
o 231mg DTT (154.25g/mol * 0.0005mol/L * 3L)
o pH 7.6
o Fill to 3L
o Filter 2L with 0.22 m filter into 2- 1L bottles
o Add 11.68g NaCl to remaining 1L (58.44g/mol * 0.2mol/L * 1L)
o Filter salt buffer into 1L bottle
o Degas buffers
o Add 100L saturated PMSF (in ethanol) to buffers prior to use
o If buffer is a few days old, add fresh DTT
o Things grow well in tris buffer, so buffer probably isn’t usable after a few
days, unless azide is added
 Saturated Ammonium Sulfate, 3.9 M, pH 7.0
o Prepare well in advance, as this requires substantial time and effort to
dissolve
o Add 12.9g ammonium sulfate to a 50 mL falcon tube and fill to 25 mL
with lysis buffer or 20 mM Tris/HCl buffer (larger batches are fine,
solution will keep)
o Vigorously stir and use slight heat to dissolve ammonium sulfate
o Check pH and adjust as necessary
PROTOCOL
 Add lysis buffer to 30 mL and thaw cells
 Make a few mL of saturated PMSF in ethanol (43 mg/mL)
 Add 160 L saturated PMSF to cells
 Add aliquot of non-his protease inhibitor cocktail
 Make 2 mL of 25 mg/mL lysozyme in lysis buffer (50 mg)
 Add lysozyme to cells
 Incubate on ice 20 minutes, agitating every ~5 min
 Add 80 mg solid deoxycholate to cells
 Transfer to 250 mL flask and shake at 100-150 rpm at 37C for 15 min (should be
very viscous)
 Prepare small amounts (small scoop) of DNase and RNase in small tubes and
dissolve in ~200 L lysis buffer
Klevit Lab
Amanda Clouser























2/13/16
Add DNase and RNase to cells
Add MgCl2 to 10 mM to cells, 61mg for 30 mL (30mL * 10mM *
203.31mg/mmol)
Continue shaking at 100-150 rpm at 37C for 15 min (viscosity should decrease
substantially)
Take 20 L sample for SDS-PAGE and store at -20C (same for all samples)
Transfer lysate to SS34 tubes and centrifuge 15 min at 15,000 rpm at 4C
Take 20 L samples of supernatant and pellet (resuspended in 30 mL buffer)
Transfer supernatant to fresh 250 mL flask and place on slow shaker at RT
Add drop-wise saturated ammonium sulfate (3.9 M) in buffer (lysis buffer
preferred, or some other Tris/HCl buffer, pH 8.0) to 40% saturation, or 20 mL to
30 mL lysate, over the course of a few minutes
Continue stirring at RT for 30 min
(Take 20 L lysate sample for SDS-PAGE) Recommended for new protein
Centrifuge 10 min at 15,000 rpm at 20C
(Take 20 L sample of supernatant for SDS-PAGE and discard supernatant)
Dissolve pellet in 30 mL “HSP27 Buffer 1”
Add additional 60 L PMSF to dissolved pellet
(Take 20 L sample for SDS-PAGE)
Run sample over G-25 column (desalting) freshly equilibrated with HSP27 Buffer
1 with fresh 100 L PMSF (1L)
o Purge line A on Captain with buffer
o Equilibrate- method “G25eq” under Ying (start this at least an hour ahead
of loading)
o Load superloop with sample
o Run- method “G25run” under Ying
Pool fractions generously where protein elutes in SS34 tubes
Add PMSF to sample (~1 L / mL)
(Take 20 L sample for SDS-PAGE)
Centrifuge 10 min at 15,000 rpm at 20C
Take 20 L samples of supernatant (and pellet (redissolved in same volume) for
SDS-PAGE)
Run supernatant over DEAE column (anion exchange) freshly equilibrated with
HSP27 Buffer 1 with fresh 100 L PMSF (1L)
o Purge line A with buffer and line B with buffer + 200 mM NaCl on
Rosalind
o Equilibrate manually (start this at least two hours before loading)
o Load superloop with sample
o Run- method “ajb Hsp27 FL DEAE voller test” (4 hours total, protein
should appear in 2-3 hours)
Take 5 L samples of fractions in major peaks as well as 1-2 from each small,
early, or late peaks for SDS-PAGE
o It is preferable to analyze the fractions by SDS-PAGE before pooling to
check purity, but pooling based on A280 chromatogram is satisfactory if
time is lacking. Always analyze fractions later in that case.
Klevit Lab
Amanda Clouser




2/13/16
Pool fractions into two tubes, separating main peak and tail/second peak
Add PMSF to samples (~2 L / mL)
Add solid ammonium sulfate to each sample to final saturation of 50% (1.95 M),
or 5.15g per 20 mL (132.14g/mol * 1.95M * 0.020L)
Nutate overnight at 4C
DAY 2
MATERIALS
 Prepare 4L phosphate buffer (50 mM NaPi, 100 mM NaCl, 0.5 mM DTT, 0.5 mM
EDTA, pH 7.5, PMSF)
o 22.72g dibasic sodium phosphate, anhydrous (142g/mol)
o 4.8g monobasic sodium phosphate, anhydrous (120g/mol)
o 23.4g NaCl (58.44g/mol * 0.1mol/L * 4L)
o 309mg DTT (154.25g/mol * 0.0005mol/L * 4L)
o 5mL 0.4M EDTA (4L * 0.0005M / 0.4M)
o 800 L saturated (43 mg/mL) PMSF
o pH to 7.5
o Filter with 0.22 M filter into separate 2L bottles
o Degas one 2L bottle for SEC
PROTOCOL
 Centrifuge nutated samples for 10 min at 15,000 rpm at 20C
 (Take 20 L sample of supernatants for SDS-PAGE and discard supernatants)
 Redissolve pellets in 2 mL phosphate buffer or less, with fresh PMSF (~1 L /
mL) by swirling or gentle pipetting
 (Take 5 L samples for SDS-PAGE)
 Place samples in 10,000 Da MW dialysis cassettes
 Dialyze in 2 sets of at least 1L phosphate buffer (fresh PMSF) at 4C over the
course of 3-4 hours (mediocre dialysis is ok, as you’re running the sample over a
column with the same buffer)
 Remove samples from dialysis and centrifuge 10 min at 15,000 rpm at 20C
 Take 5 L samples for SDS-PAGE
 Run supernatant over sdx200 column (size exclusion) freshly equilibrated with
phosphate buffer
o Clean 2 mL loop with buffer
o Purge line A with buffer
o Equilibrate manually (start this at least two hours before loading) with 2
CV of buffer at 1 mL/min
o Inject sample
o Run- method sdx200 under Scott
 Take 5 L samples of fractions in major peaks as well as 1-2 from each small,
early, or late peaks for SDS-PAGE
Klevit Lab
Amanda Clouser



2/13/16
o It is preferable to analyze the fractions by SDS-PAGE before pooling to
check purity, but pooling based on A280 chromatogram is satisfactory if
time is lacking. Always analyze fractions later in that case.
Pool fractions
Add solid ammonium sulfate to each sample to final saturation of 50% (1.95 M),
or 5.15g per 20 mL (132.14g/mol * 1.95M * 0.020L)
Nutate overnight at 4C
DAY 3
MATERIALS
 Prepare 2L phosphate buffer (50 mM NaPi, 100 mM NaCl, 0.5 mM DTT, 0.5 mM
EDTA, pH 7.5, PMSF)
o 11.36g dibasic sodium phosphate, anhydrous (142g/mol)
o 2.4g monobasic sodium phosphate, anhydrous (120g/mol)
o 11.7g NaCl (58.44g/mol * 0.1mol/L * 4L)
o 155mg DTT (154.25g/mol * 0.0005mol/L * 4L)
o 2.5mL 0.4M EDTA (4L * 0.0005M / 0.4M)
o 400 L saturated (43 mg/mL) PMSF
o pH to 7.5
o Filter with 0.22 M filter
PROTOCOL
 Centrifuge nutated samples for 10 min at 15,000 rpm at 20C
 (Take 20 L sample of supernatants for SDS-PAGE and discard supernatants)
 Redissolve pellets in 2 mL phosphate buffer or less, with fresh PMSF (~1 L /
mL) by swirling or gentle pipetting
 (Take 5 L samples for SDS-PAGE)
 Place samples in 10,000 Da MW dialysis cassettes
 Dialyze in 2 sets of at least 1L phosphate buffer (fresh PMSF) at 4C over the
course of 4-6 hours
 Remove samples from dialysis and centrifuge 10 min at 15,000 rpm at 20C
 Take 5 L samples for SDS-PAGE
 Add 10 L to 140 L buffer and measure A280, with buffer blank! (DTT can
interfere with signal)
o Extinction coefficient = 40,450M-1cm-1
o MW = 22,867g/mol
o C (molar) = A280 * 15 / (40,450M-1cm-1 * 1cm)
o C (mg/mL) = C (molar) * 22,867g/mol * V
 Aliquot supernatants into labeled tubes for storage
 Flash freeze in liquid nitrogen
 Store at -80C
 Analyze SDS-PAGE samples with 15% acrylamide gels
Klevit Lab
Amanda Clouser
2/13/16
o Try to maintain similar ratios across all samples, for example, use 1 L
per 1 mL of supernatant or pellet that it came from, add water to 5 L, add
5 L load dye, and load 5-10 L
Klevit Lab