Protein Production in Plate Format

2015-01-13

azim58 - Protein Production in Plate Format


Protein Production in Plate Format
Miscellaneous Notes About Protein Production

Materials
Note that this spreadsheet could potentially be modified so that some of
the Biomek tips are reused rather than new ones used for each step. In
order to do this, which tip box is used with which culture plate should
be recorded.
Materials for Protein Production in Plate 4-11-12.xlsx
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-24-2014d0911\01-24-2014d0911\Materials for Protein Production in Plate 4-11-12.xlsx"
200 uL pipette tips for removing supernatent soluble protein (1 box per
plate) <- make sure you have enough of these
10 mM IPTG

o 10*mmol/(1*L)*1*mol/(1000*mmol)*238.3*g/(1*mol)*1000*mg/(1*g)*0.04*
L = 95.52 mg IPTG in 50 mL H20

Lysis Buffer

o 9.85 mL 1X PBS
o 1% triton X-100 (so 100 uL)
- last seen on Felicia's shelf

o 1 mM PMSF (a protease inhibitor) (so 50 uL of 0.2 M PMSF
- 0.348g into 10mL of isopropanol
- last seen DNase box 3rd shelf in -20 C (powder last seen in
back chemical cabinet under "P")
- PMSF stands for phenylmethanesulfonylfluoride or
phenylmethylsulfonyl fluoride, and it is a serine protease inhibitor

o 1 complete protease inhibitor tablet (add tablet just before use)
- last seen at 4 C fridge by door on JC's shelf
- official name: Complete Protease Inhibitor Cocktail from Roche


Lysozyme

o last seen 4 C fridge
o final concentration
(10*"mg"/(5000*"uL")*2.5*"uL")/(100*"uL")*1000*"ug"/(1*"mg") = 0.05
ug/uL

DNAse and MgCl2

o 200 ug/mL prepared with 1 mg in 5 mL RNAse free H20
- last seen -20 C in 15 mL tube

o 2.5 ug/mL prepared with 375 uL of 200 ug/mL in 30 mL H20
o powder last seen -20 C Q freezer in door in small bottle

o 1 M liquid solution last seen on Felicia's shelf
o comes as MgCl2*6H20 which is 203.3 g/mol (powder last seen on
chemical shelf in back)
- Example calculation:
10*mmol/(1*L)*1*L/(1000*mL)*15*mL*1*mol/(1000*mmol)*203.3*g/(1*mol)
= 0.030 g

o last seen -20 in 15 mL tube by DNAse)

o 0.2 ug/mL DNAse and 11.25 mM MgCl2
- MgCl2
@ y*10/112.5 = 1 mM -> y = 11.25 mM
@ 1000*mM*y/30*mL = 11.25 mM -> y = 0.338 mL or 338 uL

- DNAse
@ 200*ug/mL*y/(30000*uL) = 0.2*ug/mL -> y = 30 uL

- 10 uL added to each well
@ final conc. MgCl2: 10*"uL"*11.25*"mM"/(100*"uL")= 1.1 mM
@ final conc. DNAse:
10*"uL"*0.2*"ug"/"mL"/(100*"uL")*1*"mL"/(1000*"ug")= 0.02 ug/mL

- prepared by adding 0.6 uL of 200 ug/mL DNAse to 30 mL H20 and
338 uL 1 M MgCl2 to the same 30 mL.


Plates (96 well low profile storage plate) (make sure to have plenty or
extra of this type of plate since they hold plenty of volume, are still
reasonably small, and don't break in the centrifuge) (note that these
don't break as easily during centrifugation) (2X this type of plate for
overnight culture, and protein production which is the same plate used
for the insoluble protein lysate at the end of the protocol)
Plates (Plain 96 well round bottom assay plates) (round bottom plates can
be ordered from Denville scientific (cat no P9735)
(1X this type of plate for the final soluble protein lysate)
Plates (384 well plates for printing) (1X this type of plate for printing)

Airpore tapesheets

set arfter IPTG has been added
3M tapesheets

has been added
Polystyrene reagent reservoir
LB/carb

EDTA

Biomek Tips

o 4 per batch and 2 per plate
- 1/10 dilution and glycerol stock (per plate and 2 per batch)
- IPTG plates (per batch)
- transfer to deep well plates and add lysis buffer (per plate)
- add DNAse/MgCl2 (per batch)


o 2 per batch
- add lysozyme (per batch)
- add EDTA (per batch)


Glycerol


Protocols

square storage culture plate
be good idea to perform this incubation as well as other incubations
with the HiGro. May want to use 200 rpm instead of 250 rpm with the
HiGro. If the HiGro is used, make sure to clean afterward by spraying
with 70% ethanol and drying.
which samples if any.
o 1/100 dilution may be best. This is the dilution they do in the
Molecular Cloning book protocol.
- took about 110 min to reach OD between 0.5-1.0 with this
dilution last time. 1/10 dilutions have taken about 30 min in the
past.

o a 1/100 dilution can be made by diluting 10 uL culture into 1000
uL LB

glycerol stock is probably appropriate)
o Ex calculation for adding glycerol to all 200 uL:
40%*y/(200*uL+y)=25% -> y = 333.3 uL so add 333.3 uL 40% glycerol to
200 uL culture

2 hr depending on dilution)
in a glass container since the glass will probably break.
IPTG. Alternatively, plate manipulations can be performed with the
Biomek.
o It may be best to try a range of IPTG concentrations 0.01-5 mM.
- 11.1 uL (per 100 uL) of 10 mM IPTG for a final concentration
of 1 mM IPTG.

o Make sure to return IPTG to -20 C

o It may be good to try a range of times (1, 2, 4, 6 hrs). 4 hr
seems to work well for many situations
o It may be good to try a range of temperatures (15-42 C). 37 C
seems to work well for many situations.
o Note that different media can be tried as well and that pH can
also affect expression (Molecular Cloning book section 15.19)

to measure the OD
regular 96 well ELISA assay plates tend to break at this centrifuge
speed. Therefore, the sample should be transferred to 96 well PCR
plates with pointed wells (it may be harder to see and remove the
supernatent from the pellet), or into the 2 mL volume 96 well Plates
for growing cultures, or the 96 well low profile storage plate.
o Be careful not to add too much lysozyme. See Notes about too much
lysozyme during plate production

o Seems to take about 3-4 hr for a whole batch of plates to thaw,
and about 1 hr 30 min for a whole batch to freeze.

may be a good idea to thaw at 4 C so that the protein is not sitting
around too long at room temperature
o To keep track of freeze thaw cycles: F T F T F T

o see Materials section above for preparation of solution
o It may be best to adjust the depth of the Biomek addition so that
the liquid from the tips just barely touches the liquid in the wells.

rotator in 4 C room. Plates may need to be taped together.
o a 384 well plate may be necessary for slide printing; need at
least 20-30 uL volume
o may want to add EDTA to prevent bacterial growth. Final
concentration (1 mM).
- Make a 1/10 dilution of 500 mM EDTA for 50 mM
- 50 mM*y/(120+y) = 1 mM -> y=2.4 uL of 50 mM EDTA to 120 uL
for 1 mM

o may want to add glycerol to slow down denaturation during
freezing and thawing process in the future.
- example glycerol addition: 40%*y/(120*uL) = 2% -> y = 6 uL of
40% glycerol to about 120 uL
- example glycerol addition 2: 3 uL of 15.3% glycerol to 20 uL
for a final of 2% glycerol (15.3*3/(23) = 2)

o Note that for checking for presence of protein in a Commassie,
silver stain, or Western, it may be a good idea to load "0.15 OD
units" measured from harvesting or 40 ug of suspension into gel
o If you wish to denature the proteins, add SDS (final
concentration of 1%) and heat at 60 C 1 hr.
o Store plate at -20 C or -80 C for long term storage. Plates may
be stored at 4 C around printing time (try not to let it sit at 4 C
for longer than 1 week). When storing plates at -20 C or -80 C it is
necessary to use aluminum foil plate sealers because the clear plate
sealers just come off. Keep in mind that the more times the plates
undergo freezing and thawing the more that proteins may unfold and
aggregate.
- When replacing the plate sealer lids, there may be
condensation on the lid. Centrifuging the plates at 3000 rpm for 5
min will remove this condensation and prevent loss of sample.



Note: A diagram can be found on 6-20-11 of Notebook
"C:\kurt\storage\CIM Research Folder\DR\2013\2-19-13\protein production
diagram\scan of protein production diagram from 6-20-11 of notebook.pdf"



see also
Felicia's notes on protein production
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2013\12-2-13\Protein purification protocol 12-8-2011.xls"