6His Purification- Batch Binding
Buffers
0.5M phosphate, pH 8.0 500 ml
Na-phosphate dibasic (141.96) 35.5 g
pH with phosphoric acid
0.5 M phosphate, pH 6.0 250 ml
Na-phosphate dibasic (141.96) 8.88 g
Na-phosphate monobasic (119.96) 7.5 g
pH with phosphoric acid
Lysis Buffer (50 mM Pi, pH 8.0; 300 mM NaCl; 2M Urea; 0.1 % Tween 20; 10 mM Imidazole +
1 mM PMSF+ 1 mM Benz-HCl + 0.5 mg/ml lysozyme)
100 ml
0.5 M phosphate, pH 8.0 10 ml
NaCl (58.44) 1.75 g
Urea (60.06) 12.01 g
Tween 20 100 μl
Imidazole (68.08) 68 mg
Column Buffer (50 mM Pi, pH 8.0; 500 mM NaCl; 0.1 % Tween 20; 10 mM Imidazole; 5 mM 2-
ME + 0.1 mM PMSF+ 0.1 μg/ml PI cocktail + 0.02 % Na-azide)
500 ml
0.5 M phosphate, pH 8.0 50 ml
NaCl (58.44) 14.61 g
Tween 20 500 μl
Imidazole (68.08) 0.34 g
2-ME 175 μl
Column Equillibration Buffer
Column Buffer + 2M Urea. 6g urea/ 50 ml buffer
pH 6.0 Wash Buffer (50 mM Pi, pH 6.0; 500 mM NaCl; 5 mM 2-ME + 0.1 mM PMSF+ 0.1
μg/ml PI cocktail + 0.02 % Na-azide)
50 ml
0.5 M phosphate, pH 6.0 5 ml
NaCl (58.44) 1.46 g
2-ME 17.5 μl
Elution Buffer (50 mM Pi, pH 7.2; 500 mM NaCl; 5 mM 2-ME; 250 mM Imidazole + 0.02 %
Na-azide)
50 ml
0.5 M phosphate, pH 7.2 5 ml
NaCl (58.44) 1.46 g
2-ME 17.5 μl
Imidazole (68.08) 0.85 g
Readjust the pH because the imidazole affects it.
Expression of 6-His Fusion Proteins
1. Grow ON of single colony in 25 ml of LB-AMP at 37 °C.
2. In morning, dilute 1/50 into 250 ml fresh LB-AMP and grow at 37 °C.
3. Grow to previously determined optimal OD 600 for expression.
4. When it reaches the correct OD, save 1 ml of the culture on ice (=uninduced sample).
5. To the rest of the culture, add IPTG to desired final to induce expression.
6. Grow for an additional 2 to 4 hrs at whatever temperature you determined optimal for
expression.
7. Read OD 600 of the induced cultures.
8. Save 1 ml of the induced cultures (=induced sample).
9. Run out samples on SDS-PAGE running about 5-8 μl/lane and look for bands in the induced
culture relative to the uninduced culture. You will want to make a guesstimate of how much
protein that you expressed. Use the judgement that the standards contain 0.2 to 0.5 μg/band
depending on how much you loaded.
10. Pellet rest of culture and resuspend in about 10 ml of lysis buffer/g pellet weight. Freeze in
liquid nitrogen and store at –80 °C.
Preparation of Resin
1. The resin comes as a 50% slurry and it will have to be washed and equillibrated.
2. The binding capacity of the resin is approximately 5-10 mg protein/ml of packed resin. Do
not use any more resin than necessary as it will increase the background substantially.
3. Take the appropriate amount of resin and wash 2Xs in 10 volumes of elution buffer. To
wash resin, resuspend in wash buffer and incubate for 5-10’ and then pellet for 3-4’ at speed
5 on a clinical.
4. Wash resin 2Xs in 10 volumes of column equilibration buffer. After spinning, remove
excess buffer. Resin is ready to go. Keep on ice.
Preparation of Lysate
(Keep everything as cold as possible by always having samples immersed in an ice bucket.
Make sure that samples remain in ice bucket during sonication because this generates a lot of
heat).
1. To prepare lysate, thaw cell suspension by immersing the ziplock bag or conical tube in cool
water. Mix often to insure even thawing and avoid ice chunks or warm spots.
2. Transfer cell suspension to a small plastic beaker- it will be quite goopy from the DNA at
this point. Glass is supposed to be better for sonication, but I’m always afraid that it will
crack. Sonicate at least 2 X 20 sec with a medium to large tip sonicator. You want to set the
sonicator at a setting that gives good sonication but avoids frothing. Monitor extent of
sonication by reduction in the amount of goopy DNA- the solution should become quite
liquid. Save a 25 μl aliquot of this sample as lysate.
3. Add 2-ME to 5 mM and bring NaCl to 0.5 M. Centrifuge at 18K for 20’ in JS-25.5 rotor.
4. Remove sup to a fresh tube and save a 25 μl aliquot of this sample as column load.
Running Column
(Note- from this point onward, you need to take extra precautions that everything stays cold so
you will be in the coldroom as often as possible instead of just keeping the tubes in an
icebucket).
It is simplest to actually batch bind the protein to the resin and pour it into a column and
do the washes in column format, and I usually do it this way. I do then pour it into a column and
do the last washes and elution in column format- I often find that you get better elution from a
column than in batch. Alternatively, you can run the entire procedure like a classical column,
but this takes longer. All of the column steps should be done in the cold room to keep everything
cold. Also, I don’t set up any fancy parastaltic pumps- I just let the columns flow by gravity.
1. Take the desired amount of prepared Ni-NTA agarose in a 15 or 50 ml conical depending on
volume of lysate.
2. Add the clarified lysate to the resin and batch bind at least 1 to 2 hrs or ON at 4 °C. If you
batch bind ON then I sometimes add NaN3 to 0.02% to prevent growth in the extract which is
very high in protein and other goodies.
3. Pour the resin into a column and let the resin settle into the bottom of the column.
4. Wash out the 50 ml conical with 10 volumes of column equilibration buffer.
5. Wash resin with 10 volumes of column buffer.
6. Wash resin with 10 volumes of pH 6.0 wash buffer.
7. Stop flow of the column when the last of pH 6.0 has just entered the bed of the resin so there
is no buffer sitting on top of the resin bed.
8. Gently pipet on a small volume (1-2 ml) of elution buffer onto the surface of the resin. Begin
to flow the column and collect 1 1 ml fraction. What this does is basically let the elution
buffer enter into the resin of the column. Let sit 30-60’ at 4 °C.
9. Begin eluting column. Collect 10-15 1 ml fractions depending on the size of the column.
The protein elution pattern varies a lot with Ni-NTA resin and will probably need to be
optimized.
10. To see the elution profile, you can do a nitrocellulose spot test. Take a small piece of NC
paper and number for column fractions. Spot 1 μl of each fraction on the paper, and let it air
dry. Then stain the NC paper with Ponceau S and destain in 5% acetic acid as if you were
doing a western blot. The protein containing fractions will turn bright pink which the other
fractions will be blank.
11. Run out all protein containing fractions on SDS-PAGE and coomassie stain to determine
purity of protein. Generally you should get fairly pure protein with just some lower bands
which are either proteolysis or inefficient translation.
12. Pool all fractions that contain sufficient protein and dialyze against your desired buffer. For
injection into rabbits, I’ve found that 50 mM MOPS, pH 7.2; 50 mM NaCl; 0.1 mM EGTA;
0.1 mM EDTA works well. For frog extract experiments, dialyze into 10 mM Hepes, pH
7.2; 100 mM KCl; 25 mM NaCl; 50 mM sucrose; 0.1 mM EDTA; 0.1 mM EGTA. There
may be some precipitation after the dialysis. For the immunogen, this is fine- a few solids
are good for the immune system. For extract experiments, precipitation is bad so the final
sample should be centrifuged and then filtered thru a 0.2 um filter before use. Samples can
be stored at 4 °C short-term and in aliquots at –80 for longer term storage.
13. To determine concentration, use Bradford with BSA as a standard. Alternatively, you can
use the Von-Hippel equation to calculate the molar extinction coefficient for a given protein.
When measuring A280, a 1 mg/ml solution has an OD equivalent to the ((#trpX5690 +
#tyrX1280)/ MW of the protein) X 1.05.
0.5M phosphate, pH 8.0 500 ml
Na-phosphate dibasic (141.96) 35.5 g
pH with phosphoric acid
0.5 M phosphate, pH 6.0 250 ml
Na-phosphate dibasic (141.96) 8.88 g
Na-phosphate monobasic (119.96) 7.5 g
pH with phosphoric acid
Lysis Buffer (50 mM Pi, pH 8.0; 300 mM NaCl; 2M Urea; 0.1 % Tween 20; 10 mM Imidazole +
1 mM PMSF+ 1 mM Benz-HCl + 0.5 mg/ml lysozyme)
100 ml
0.5 M phosphate, pH 8.0 10 ml
NaCl (58.44) 1.75 g
Urea (60.06) 12.01 g
Tween 20 100 μl
Imidazole (68.08) 68 mg
Column Buffer (50 mM Pi, pH 8.0; 500 mM NaCl; 0.1 % Tween 20; 10 mM Imidazole; 5 mM 2-
ME + 0.1 mM PMSF+ 0.1 μg/ml PI cocktail + 0.02 % Na-azide)
500 ml
0.5 M phosphate, pH 8.0 50 ml
NaCl (58.44) 14.61 g
Tween 20 500 μl
Imidazole (68.08) 0.34 g
2-ME 175 μl
Column Equillibration Buffer
Column Buffer + 2M Urea. 6g urea/ 50 ml buffer
pH 6.0 Wash Buffer (50 mM Pi, pH 6.0; 500 mM NaCl; 5 mM 2-ME + 0.1 mM PMSF+ 0.1
μg/ml PI cocktail + 0.02 % Na-azide)
50 ml
0.5 M phosphate, pH 6.0 5 ml
NaCl (58.44) 1.46 g
2-ME 17.5 μl
Elution Buffer (50 mM Pi, pH 7.2; 500 mM NaCl; 5 mM 2-ME; 250 mM Imidazole + 0.02 %
Na-azide)
50 ml
0.5 M phosphate, pH 7.2 5 ml
NaCl (58.44) 1.46 g
2-ME 17.5 μl
Imidazole (68.08) 0.85 g
Readjust the pH because the imidazole affects it.
Expression of 6-His Fusion Proteins
1. Grow ON of single colony in 25 ml of LB-AMP at 37 °C.
2. In morning, dilute 1/50 into 250 ml fresh LB-AMP and grow at 37 °C.
3. Grow to previously determined optimal OD 600 for expression.
4. When it reaches the correct OD, save 1 ml of the culture on ice (=uninduced sample).
5. To the rest of the culture, add IPTG to desired final to induce expression.
6. Grow for an additional 2 to 4 hrs at whatever temperature you determined optimal for
expression.
7. Read OD 600 of the induced cultures.
8. Save 1 ml of the induced cultures (=induced sample).
9. Run out samples on SDS-PAGE running about 5-8 μl/lane and look for bands in the induced
culture relative to the uninduced culture. You will want to make a guesstimate of how much
protein that you expressed. Use the judgement that the standards contain 0.2 to 0.5 μg/band
depending on how much you loaded.
10. Pellet rest of culture and resuspend in about 10 ml of lysis buffer/g pellet weight. Freeze in
liquid nitrogen and store at –80 °C.
Preparation of Resin
1. The resin comes as a 50% slurry and it will have to be washed and equillibrated.
2. The binding capacity of the resin is approximately 5-10 mg protein/ml of packed resin. Do
not use any more resin than necessary as it will increase the background substantially.
3. Take the appropriate amount of resin and wash 2Xs in 10 volumes of elution buffer. To
wash resin, resuspend in wash buffer and incubate for 5-10’ and then pellet for 3-4’ at speed
5 on a clinical.
4. Wash resin 2Xs in 10 volumes of column equilibration buffer. After spinning, remove
excess buffer. Resin is ready to go. Keep on ice.
Preparation of Lysate
(Keep everything as cold as possible by always having samples immersed in an ice bucket.
Make sure that samples remain in ice bucket during sonication because this generates a lot of
heat).
1. To prepare lysate, thaw cell suspension by immersing the ziplock bag or conical tube in cool
water. Mix often to insure even thawing and avoid ice chunks or warm spots.
2. Transfer cell suspension to a small plastic beaker- it will be quite goopy from the DNA at
this point. Glass is supposed to be better for sonication, but I’m always afraid that it will
crack. Sonicate at least 2 X 20 sec with a medium to large tip sonicator. You want to set the
sonicator at a setting that gives good sonication but avoids frothing. Monitor extent of
sonication by reduction in the amount of goopy DNA- the solution should become quite
liquid. Save a 25 μl aliquot of this sample as lysate.
3. Add 2-ME to 5 mM and bring NaCl to 0.5 M. Centrifuge at 18K for 20’ in JS-25.5 rotor.
4. Remove sup to a fresh tube and save a 25 μl aliquot of this sample as column load.
Running Column
(Note- from this point onward, you need to take extra precautions that everything stays cold so
you will be in the coldroom as often as possible instead of just keeping the tubes in an
icebucket).
It is simplest to actually batch bind the protein to the resin and pour it into a column and
do the washes in column format, and I usually do it this way. I do then pour it into a column and
do the last washes and elution in column format- I often find that you get better elution from a
column than in batch. Alternatively, you can run the entire procedure like a classical column,
but this takes longer. All of the column steps should be done in the cold room to keep everything
cold. Also, I don’t set up any fancy parastaltic pumps- I just let the columns flow by gravity.
1. Take the desired amount of prepared Ni-NTA agarose in a 15 or 50 ml conical depending on
volume of lysate.
2. Add the clarified lysate to the resin and batch bind at least 1 to 2 hrs or ON at 4 °C. If you
batch bind ON then I sometimes add NaN3 to 0.02% to prevent growth in the extract which is
very high in protein and other goodies.
3. Pour the resin into a column and let the resin settle into the bottom of the column.
4. Wash out the 50 ml conical with 10 volumes of column equilibration buffer.
5. Wash resin with 10 volumes of column buffer.
6. Wash resin with 10 volumes of pH 6.0 wash buffer.
7. Stop flow of the column when the last of pH 6.0 has just entered the bed of the resin so there
is no buffer sitting on top of the resin bed.
8. Gently pipet on a small volume (1-2 ml) of elution buffer onto the surface of the resin. Begin
to flow the column and collect 1 1 ml fraction. What this does is basically let the elution
buffer enter into the resin of the column. Let sit 30-60’ at 4 °C.
9. Begin eluting column. Collect 10-15 1 ml fractions depending on the size of the column.
The protein elution pattern varies a lot with Ni-NTA resin and will probably need to be
optimized.
10. To see the elution profile, you can do a nitrocellulose spot test. Take a small piece of NC
paper and number for column fractions. Spot 1 μl of each fraction on the paper, and let it air
dry. Then stain the NC paper with Ponceau S and destain in 5% acetic acid as if you were
doing a western blot. The protein containing fractions will turn bright pink which the other
fractions will be blank.
11. Run out all protein containing fractions on SDS-PAGE and coomassie stain to determine
purity of protein. Generally you should get fairly pure protein with just some lower bands
which are either proteolysis or inefficient translation.
12. Pool all fractions that contain sufficient protein and dialyze against your desired buffer. For
injection into rabbits, I’ve found that 50 mM MOPS, pH 7.2; 50 mM NaCl; 0.1 mM EGTA;
0.1 mM EDTA works well. For frog extract experiments, dialyze into 10 mM Hepes, pH
7.2; 100 mM KCl; 25 mM NaCl; 50 mM sucrose; 0.1 mM EDTA; 0.1 mM EGTA. There
may be some precipitation after the dialysis. For the immunogen, this is fine- a few solids
are good for the immune system. For extract experiments, precipitation is bad so the final
sample should be centrifuged and then filtered thru a 0.2 um filter before use. Samples can
be stored at 4 °C short-term and in aliquots at –80 for longer term storage.
13. To determine concentration, use Bradford with BSA as a standard. Alternatively, you can
use the Von-Hippel equation to calculate the molar extinction coefficient for a given protein.
When measuring A280, a 1 mg/ml solution has an OD equivalent to the ((#trpX5690 +
#tyrX1280)/ MW of the protein) X 1.05.
Comments
Post a Comment