BRCA1/BARD1 (L/L) Purification Protocol
Co-expression of “long/long” BCBD proteins
NOTE: BRCA1/BARD1 long/long degrades rapidly during purification. As a result, it is best to
finish purification as quickly as possible.
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His/Flagged-BRCA1 (Residues 1-304)
BARD1 (Residues 25-327)
ε280 = 39.89cm-1mM-1
MW = 36,098.4 Da
MW = 33,760.3 Da
[DAY 1] – PREPARE AS MUCH AS YOU CAN FOR TOMORROW AND THE DAY AFTER!
Co-Transformation: Transformation via electroporation
1. Thaw 1 aliquot of electrically competent BL21 E.coli cells on ice.
2. Add 50 uL of sterile ddH2O to cells and mix gently with the pipet.
3. Thaw pET28-BARD1 plasmid and pCOT7N-BRCA1 plasmid DNA stocks on ice.
4. Add 1 uL of each DNA plasmid stock to thawed cell aliquot.
5. Transfer cells to a clean electro-cuvette. Make sure that there are no bubbles
present in the cuvette after adding the cells, as this could effect transformation.
6. Remove lid from the cuvette and electroporate cells with the electroporator.
a. Make sure that the parameters are set to Bacteria and that the readout is set
to Time.
b. Each electroporation should give a time readout of >2.0; if less, transfer cells
to a new electrocuvette and try again.
7. Add 300 mL of sterile LB to cuvette and mix with the pipette.
8. Transfer mixture to a 1.5 mL microfuge tube and shake @ 37ºC for 20-40 minutes.
9. Plate on a Kanomycin/Chloamphenicol (Kan/Cam) plate.
10. Invert the plate and allow cells to grow @ 37ºC overnight in the warm room.
Suggestions
 Prepare and autoclave 1L LB media per 1 aliquot BL21 cells used for tomorrow.
[DAY 2] – GROWTH AND EXPRESSION
Growth and Induction – Unlabeled LB preps
1. In the morning, add antibiotics to LB culture flask(s).
a. For 1L LB, add 2 mL 1000X Kan and 1 mL 1000X Cam
2. Remove culture plate from 37ºC warm room and verify that there is growth present.
3. Add ~1.5 mL sterile ddH2O to culture plate and resuspend cells w/ a flame sterilized
glass stir rod.
4. Transfer ~1 mL resuspended cells to LB +kan/cam culture flask.
5. When A600 reaches ~0.3, add ZnCl2 to final concentration of 100-200 uM ( ~1 mL of
200 mM stock to 1L LB)
6. Continue shaking at 37ºC until reaching an A600 of ~0.4-0.6.
7. Take 1 mL pre-induction sample for SDS-PAGE gels.
8. Induce each culture flask dry IPTG to 1 mM.
9. Let BRCA1/BARD1 expression continue for 4 hours after induction at a continued
37ºC.
10. Take a 1 mL post-induction sample for SDS-PAGE gels.
11. To harvest, transfer cells to 1L centrifuge bottles.
12. Spin in centrifuge at 6000g for 15-25 minutes.
13. Pour off supernatant.
14. Resuspend cells in 10-15 mL Nickel Start Buffer for each 1L of culture.
a. 25 mM Tris-HCl, 200 mM NaCl, 10 mM Imidazole, pH 7.6
i. Note: Total volume of resuspended cells should be around 30 mL for 23L of culture.
15. Store in -80ºC freezer until you are ready to lyse cells and purify BRCA1/BARD1.
Suggestions
 Equilibrate SDX200 column in 25mM NaPi, 150 mM NaCl, pH 7.0 overnight for use
tomorrow.
 If you do not have previously prepared nickel column(s), be sure to do so via the
protocol below at least one day prior to purification.
Nickel Column Preparation (in case you do not have any premade Nickel Columns)
1. Measure out the appropriate amount of Ni resin/20% EtOH into a 50mL conical
tube.
a. 3 mL resin per 1L culture, 5 mL resin for 2-3L culture, and 7-10 mL for larger
preps.
2. Fill the conical tube w/ ddH2O and centrifuge at full speed in a table top centrifuge
for 5 minutes. Decant carefully.
3. Add 0.5-1.0 CV of 100mM NiSO4 solution to resin and nutate for 5 minutes.
4. Fill tube with water, resuspend the resin and spin for 5 minutes. Decant.
5. Wash resin 3 more times with as much water as fits in the tube. Resuspend resin,
spin, and decant.
6. Wash resin a final time in Ni Start buffer (25 mM Tris-HCl, 200mM NaCl, 10 mM
Imidazole, pH 7.6). Resuspend, spin, decant.
7. Resin is now ready to use. Resuspend in a small volume of start buffer to transfer
the resin out of the tube if necessary or supernatant can be directly added to resin.
a. Alternatively, columns can be stored in 20% EtOH after washing with ddH2O
for long-term storage.
[DAY 3] – PROTEIN PURIFICATION – MUST BE COMPLETED TODAY!
1. Thaw previously frozen BRCA1/BARD1 containing cells on ice.
2. Make 1 mL PMSF (43mg/mL EtOH).
3. Lyse resuspended cells via French Press twice.
a. Add 50 uL PMSF sol’n prior to the first press.
b. Add another 50 uL PMSF sol’n after the first and second presses.
4. Add 1 aliquot (~200uL) protease inhibitor cocktail for His-tagged proteins (this PI
does not contain EDTA).
5. Add 200 uL 1M MgCl2 to the pressed cells.
6. Add a small amount of DNase and RNase to pressed cells; mix by inversion and let
stand on ice for ~30 minutes.
7. Equilibrate Nickel column resin while the DNase and RNase digestion is proceeding.
a. If your Ni column(s) are currently stored in 20% EtOH, elute off EtOH w/ 1-2
CV ddH2O.
b. Subsequently wash column(s) with 2-3 CV Ni Start Buffer.
c. Column is now equilibrated.
8. Once DNA and RNA digestion is complete, spin the cell lysate at 13k rpm (20000g)
for 30 minutes in an SS-34 rotor.
9. Save a small aliquot of supernatant for SDS gels. Add the remaining supernatant to
the Ni resin while in a cold room (4ºC).
10. Run the Ni Column
a. Allow supernatant to elute from column and collect the flow through (FT).
Collect a sample for SDS-PAGE gels.
b. Wash with 10CV of each buffer and collect washes for later SDS-gel analysis.
ii. Start buffer (10 mM imidazole)
iii. 20 mM imidazole
iv. 50 mM imidazole
v. Elute with 500 mM imidazole
11. Concentrate elution using the 15 mL/5k MWCO concentrators. Final volume
depends on the gel filtration column, but it is good to concentrate BRCA1/BARD1 to
between 1 and 1.5 mL for an SDX200 column.
12. Run sizing column (it was equilibrated last night, right?!). Use SDX200 column; use a
smaller column for smaller preps, and a larger column for larger preps.
a. Buffer: 25 mM NaPi, 150 mM NaCl, ph 7.0
b. Use appropriate column method.
13. Add 1 uL PMSF to each peak-generating fraction and immediately store at 4ºC.
14. Run gel to determine which fractions contain BRCA1/BARD1.
15. Pool BRCA1/BARD1 containing fractions
16. Add 0.5 mM DTT and 0.4 mM PMSF sol’n.
17. Concentrate using the 15 mL/5K MWCO to approximately 1 mM.
18. Flash-freeze protein and store in -80C freezer.