M
Mini Expression
-Grow up 5 ml o/n culture (w/ Ampicillin)
-Inoculate fresh 5 ml LB-Amp with 50 µl o/n culture.
-Incubate 2-3 h with shaking at 37°C.
-Add 2 µl IPTG (0.2 mM)
-Grow 3-4 h with shaking at 37°C.
-Spin down 1.5 ml at 13,000 rpm.
-Add 150 µl PBS + 50 µl of 4X reducing buffer (SDS-PAGE reducing buffer)
-Mix
-Boil for 10 min.
Load on SDS-PAGE gel and run.
Mitomycin C (also see T cell proliferation)
-Add 4 ml sterile PBS to Mit. C powder.
-This is now a 500 µg/ml stock.
Need 50 µg Mit C/ml of antigen presenting cells.
-Need APCs @ 5x10-7 cells/ml
-Incubate in the dark for 20 min at 37°C.
-Wash 3X with 50 ml CTL media before adding to T cells.
MTT Assay (also see T cell proliferation)
Materials:
Use Thiazolyl Blue Tetrazolium Bromide (Sigma M2128) @ 5 mg/ml in sterile PBS.
10 ml Extraction Buffer (20% SDS, 50% DMF (dimethylformamide) i.e., 5 ml H20 + 5 ml DMF + 2 g SDS.
1-Add 25 µl Thiazolyl Blue Tetrazolium Bromide to each well (100 µl) for 2.5 h at 37°C.
2-Add 100 µl Extraction Buffer to solubilize cells and incubate o/n @ 37°C.
3-Read at 570 nm.
Mini Expression
-Grow up 5 ml o/n culture (w/ Ampicillin)
-Inoculate fresh 5 ml LB-Amp with 50 µl o/n culture.
-Incubate 2-3 h with shaking at 37°C.
-Add 2 µl IPTG (0.2 mM)
-Grow 3-4 h with shaking at 37°C.
-Spin down 1.5 ml at 13,000 rpm.
-Add 150 µl PBS + 50 µl of 4X reducing buffer (SDS-PAGE reducing buffer)
-Mix
-Boil for 10 min.
Load on SDS-PAGE gel and run.
Mitomycin C (also see T cell proliferation)
-Add 4 ml sterile PBS to Mit. C powder.
-This is now a 500 µg/ml stock.
Need 50 µg Mit C/ml of antigen presenting cells.
-Need APCs @ 5x10-7 cells/ml
-Incubate in the dark for 20 min at 37°C.
-Wash 3X with 50 ml CTL media before adding to T cells.
MTT Assay (also see T cell proliferation)
Materials:
Use Thiazolyl Blue Tetrazolium Bromide (Sigma M2128) @ 5 mg/ml in sterile PBS.
10 ml Extraction Buffer (20% SDS, 50% DMF (dimethylformamide) i.e., 5 ml H20 + 5 ml DMF + 2 g SDS.
1-Add 25 µl Thiazolyl Blue Tetrazolium Bromide to each well (100 µl) for 2.5 h at 37°C.
2-Add 100 µl Extraction Buffer to solubilize cells and incubate o/n @ 37°C.
3-Read at 570 nm.
| Molarity M.W. X Molarity =g/L µg/M.W.=µM |
N
NBT (for Western blot development)
For 5 ml:
NBT 150 mg
DMF 3.5 ml
H2O 1.5 ml
Store at -20°C, light sensitive.
Nickel Column (Phosphate Buffers)
Buffer A (10X)
20 mM Sodium Phosphate
Na2HPO4 103.5 g
NaH2PO4 15.5 g
0.5 M NaCL 88.5 g
QS to IL pH 7.4, filter
Buffer B (5X)
20 mM Sodium Phosphate
Na2HPO4 26 g
NaH2PO4 4 g
0.5 M NaCl 22.1 g
0.5 Imidazole 85 g
pH 7.4, filter
NBT (for Western blot development)
For 5 ml:
NBT 150 mg
DMF 3.5 ml
H2O 1.5 ml
Store at -20°C, light sensitive.
Nickel Column (Phosphate Buffers)
Buffer A (10X)
20 mM Sodium Phosphate
Na2HPO4 103.5 g
NaH2PO4 15.5 g
0.5 M NaCL 88.5 g
QS to IL pH 7.4, filter
Buffer B (5X)
20 mM Sodium Phosphate
Na2HPO4 26 g
NaH2PO4 4 g
0.5 M NaCl 22.1 g
0.5 Imidazole 85 g
pH 7.4, filter
Nickel Column Protein Purification Protocol (using phosphate buffers--above)
(Also see IDA Column protocol)
-Grow up corresponding clone (1 L).
-Resuspend the pellet in 20-25 ml PBS.
-Lyse using French press; spin down @ 40, 000 rpm for 20 min; filter through a 22 µM syringe
filter. Keep on ice!!!
-Save a fraction of the pellet for later analysis (if needed).
COLUMN PREPARATION (also see IDA Column protocol):
-Elute the 20% EtOH used to store 1 ml column.
-Wash with 5 vol Buffer A (i.e., 25 vol).
-Load 5 vol of 1X Ni solution (Charge Buffer if column does not come pre-charged).
-Wash excess with filtered H20.
-Equilibrate with Buffer A (5 vol).
PROTEIN PURIFICATION (also see IDA Column Protocol):
-Apply the lysate to the column (collect flow-through).
-Wash with 4% Buffer B (96% Buffer A) [Always use Buffer A to dilute B, otherwise the
phosphate buffer activity will be abolished.].
-Create your own gradient of increasing Buffer B e.g. 6--100%. Collect fractions.
-Wash the column with Strip buffer (5 vol).
-Wash with 20% EtOH and store in 20% EtOH at 4°C.
Subject respective fractions to SDS-PAGE.
Nylon Wool Purification of T cells.
1-Harvest lymph nodes and/or spleens under sterile conditions and place into incomplete CTL
media.
2-When all samples have been collected crush between two sterile, frosted slides (using the
frosted ends) over complete CTL media.
3-Filter cells and tissue through a nitex sock (or 50 ml filter top).
4-After filtering spin down @1000 RPM for 10 min @RT.
5-QS to 10 ml with complete CTL and count cells.
6-Equilibrate nylon wool (Polysciences Inc.) column with complete CTL and adjust flow so that
the drip is very slow.
7-Mix media into the nylon wool using a sterile 1 ml pipet until completely saturated and no
bubbles are present. Run 10 ml complete CTL through column and stop flow when the media
level is right above the nylon wool. Incubate at 37°C for at least 1 h. Use one 0.6 g column/1x10_8
cells.
8-Resuspend cells to purified in 1 ml complete CTL and add to the column. Add an additional 1 ml
to the tube previously containing the cells to be purified to collect all cells and add to column.
9-Start the flow very slowly and stop when the level reaches the top of the nylon wool. Add 2 ml
complete CTL. Run as above and stop flow. Add 4 ml complete CTL and incubate at 37°C for 45
min.
10-During this incubation prepare spleens for APCs.
11-Take two spleens and place them in RPMI.
12-Tease apart as above and resuspend in RBC lysing buffer (2 ml) for 2 min. Add 30 ml complete
CTL and spin down at 1500 RPM, 5 min. Depending on the appearance of the pellet this step may
have to be repeated (i.e., if too many RBCs appear to remain).
13-Carefully aspirate supernatant and wash 2X with incomplete CTL (25 ml each wash).
14-Count cells and treat with mitomycin C. Follow mitomycin C protocol.
15-Begin collecting T cells from the column. Set drip very slowly. When drops get cloudy these
should be the T cells coming off. Collect until the drops become clear again. A total of 30 ml
complete CTL will have to be added to the column to accomplish this and 30% of the total cells
added to the column originally should be recovered. Count cells.
16-Follow T cell stimulation protocol.
NaHCO3 Buffer (Sodium Bicarbonate), pH 8.6, 100 mM
8.4 g NaHCO3/L
Used for coating peptides to ELISA wells.
(Also see IDA Column protocol)
-Grow up corresponding clone (1 L).
-Resuspend the pellet in 20-25 ml PBS.
-Lyse using French press; spin down @ 40, 000 rpm for 20 min; filter through a 22 µM syringe
filter. Keep on ice!!!
-Save a fraction of the pellet for later analysis (if needed).
COLUMN PREPARATION (also see IDA Column protocol):
-Elute the 20% EtOH used to store 1 ml column.
-Wash with 5 vol Buffer A (i.e., 25 vol).
-Load 5 vol of 1X Ni solution (Charge Buffer if column does not come pre-charged).
-Wash excess with filtered H20.
-Equilibrate with Buffer A (5 vol).
PROTEIN PURIFICATION (also see IDA Column Protocol):
-Apply the lysate to the column (collect flow-through).
-Wash with 4% Buffer B (96% Buffer A) [Always use Buffer A to dilute B, otherwise the
phosphate buffer activity will be abolished.].
-Create your own gradient of increasing Buffer B e.g. 6--100%. Collect fractions.
-Wash the column with Strip buffer (5 vol).
-Wash with 20% EtOH and store in 20% EtOH at 4°C.
Subject respective fractions to SDS-PAGE.
Nylon Wool Purification of T cells.
1-Harvest lymph nodes and/or spleens under sterile conditions and place into incomplete CTL
media.
2-When all samples have been collected crush between two sterile, frosted slides (using the
frosted ends) over complete CTL media.
3-Filter cells and tissue through a nitex sock (or 50 ml filter top).
4-After filtering spin down @1000 RPM for 10 min @RT.
5-QS to 10 ml with complete CTL and count cells.
6-Equilibrate nylon wool (Polysciences Inc.) column with complete CTL and adjust flow so that
the drip is very slow.
7-Mix media into the nylon wool using a sterile 1 ml pipet until completely saturated and no
bubbles are present. Run 10 ml complete CTL through column and stop flow when the media
level is right above the nylon wool. Incubate at 37°C for at least 1 h. Use one 0.6 g column/1x10_8
cells.
8-Resuspend cells to purified in 1 ml complete CTL and add to the column. Add an additional 1 ml
to the tube previously containing the cells to be purified to collect all cells and add to column.
9-Start the flow very slowly and stop when the level reaches the top of the nylon wool. Add 2 ml
complete CTL. Run as above and stop flow. Add 4 ml complete CTL and incubate at 37°C for 45
min.
10-During this incubation prepare spleens for APCs.
11-Take two spleens and place them in RPMI.
12-Tease apart as above and resuspend in RBC lysing buffer (2 ml) for 2 min. Add 30 ml complete
CTL and spin down at 1500 RPM, 5 min. Depending on the appearance of the pellet this step may
have to be repeated (i.e., if too many RBCs appear to remain).
13-Carefully aspirate supernatant and wash 2X with incomplete CTL (25 ml each wash).
14-Count cells and treat with mitomycin C. Follow mitomycin C protocol.
15-Begin collecting T cells from the column. Set drip very slowly. When drops get cloudy these
should be the T cells coming off. Collect until the drops become clear again. A total of 30 ml
complete CTL will have to be added to the column to accomplish this and 30% of the total cells
added to the column originally should be recovered. Count cells.
16-Follow T cell stimulation protocol.
NaHCO3 Buffer (Sodium Bicarbonate), pH 8.6, 100 mM
8.4 g NaHCO3/L
Used for coating peptides to ELISA wells.
Another way of expressing concentration is called molarity. Molarity is
the number of moles of solute dissolved in one liter of solution. The
units, therefore are moles per liter, specifically it's moles of solute
per liter of solution.
molarity = moles of solute/liter of solution
Rather than writing out moles per liter, these units are abbreviated as
M or M. So when you see M or M it stands for molarity, and it means
moles per liter (not just moles).
You must be very careful to distinguish between moles and molarity.
"Moles" measures the amount or quantity of material you have;
"molarity" measures the concentration of that material. So when
you're given a problem or some information that says the
concentration of the solution is 0.1 M that means that it has 0.1 mole
for every liter of solution; it does not mean that it is 0.1 moles.
the number of moles of solute dissolved in one liter of solution. The
units, therefore are moles per liter, specifically it's moles of solute
per liter of solution.
molarity = moles of solute/liter of solution
Rather than writing out moles per liter, these units are abbreviated as
M or M. So when you see M or M it stands for molarity, and it means
moles per liter (not just moles).
You must be very careful to distinguish between moles and molarity.
"Moles" measures the amount or quantity of material you have;
"molarity" measures the concentration of that material. So when
you're given a problem or some information that says the
concentration of the solution is 0.1 M that means that it has 0.1 mole
for every liter of solution; it does not mean that it is 0.1 moles.