Preparing the Bacteria for Purification

Pre-prepared overnight cultures of bacteria were used to create bacteria with overexpressed
proteins, made possible by adding 45mL of the LB Amp+ Km+ in GST and 6xHis-USP7 and
45mL of LB Amp+ in the GST-EBNA1 and incubated for 1 hour at 37C at 250rpm. 550uL of
0.1M of IPTG was then added to the culture and incubated once more in same conditions. 150uL
of the non-induced GST-EBNA1 was added into a microfuge tube and centrifuged at 13000rpm
for 2 minutes at room temperature. This was stored at -20C. All other samples were transferred
into empty 50mL tubes.
Purification of 6xHis-USP7 using Ni2+ Affinity chromatography
Lysis/Binding Buffer was created by mixing 5.0mL of Common Buffer and 5.0uL of 5M
Imidazole and adding 1 mL to 6xHis-USP7 which was vortexed and centrifuged. 50uL of Ni2+IMAC agarose resin stock was added to the column with a yellow plug. 1mL of distilled water
(three times) and 1 mL of Lysis/Binding Buffer (three times) were all added to the column
separately, after removing the yellow plug. When the centrifugation finished, 10uL of the lysate
supernatant (USP7 Lys) was collected and store at -20C. 1mL of the crude bacterial cell lysate
was also transferred to the column. Buffer and the Elution Buffer were created by mixing 9.0mL
of Common Buffer and 27uL of Imidazole and 5.7mL of Common Buffer and 300uL of
Imidazole, respectively. The resin was then incubated. 8mL of Ni2+ Wash Buffer in 1mL volumes
was added and 200uL of Ni2+ Elution Buffer. The final unit is placed on centrifugation and stored
at -20C. The 6xHis-USP7 elution fraction was transferred to a dialysis tube in the 1xPBS pH 7.4
for 48 hours in 4C.
Purification of ENBA1 using GST Affinity Chromatography
At the same time, 1 mL of 1xPBS pH 7.4 was added to the bacterial pellet and vortexed and
centrifuged, while a pre-prepared Gluthathionine-agarose resin was washed with 1mL of distilled
water and 1mL of 1xPBS pH 7.4, each three times. Once the centrifugation was complete,
samples GST Lys and GSTE Lys were collected from the supernatants and stored in -20C. 250uL
of this lysate was added to the column, washed with 750uL of 1xPBS pH 7.4, incubated and
centrifuged. 7mL of 1xPBS pH 7.4 was added in 1mL volume intervals and after the last wash,

plugs were attached the the columns so that another wash of 1mL of 1xPBS pH 7.4 could be
suspended and samples could be collected as GST R and GSTE R, stored at -20C.
Determining the Concentration of purified 6xHis-USP7 with Bradford Assay
A Bradford Assay was conducted by mixing 975uL of 1x Bradford Reagent with 25uL of
distilled water, Ni2+ Elution Buffer and 6xHis-USP7 Elt respectively and separately. The
spectrophotometer was set at 595nm and each of the samples were measured their optical density
readings. A scatterplot was then derived in order to determine the line of best fit to determine the
protein concentration of purified 6xHis-USP7.
Preparing samples for GST Pull-Down Assay of USP7 and EBNA1
20uL of dialyzed USP7 was stored away once it finished. The columns with GST and GSTEBNA1 were washed again with 1xPBS pH 7.4 and adding around 200uL of the 6xHis-USP7
and 800uL of 1xPBS pH 7.4 to the GST and EBNA1 loaded and sealed columns for incubation.
The liquid fractions were collected and labelled as GST PD-UP and GSTE PD-UP. The
columns were then placed on centrifugation and pooled in collected tubes with the PD-UP
samples and stored at -20C. 6mL of 1xPBS pH 7.4 in 1mL intervals was added to the columns
and the final washes were collected, labelled GST-PDW and GSTE-PDW and stored at
-20C. Plugs were attached to the columns and washed with 200uL of GST Elution Buffer. Plugs
were removed after an incubation period of 10 minutes and centrifuged. The elutes were
transferred, labelled as GST PD-Elt and GSTE PD-Elt and 20uL of each were stored at
-20C.
SDS-PAGE Casting Gels
SDS-PAGE gels were made by mixing 50uL of 10% APS to the 5mL of chilled 15% Separating
Solution, along with the addition of 10uL of TEMED. This solution was added to the assembled
glass cassette in the casting apparatus with an overlay of 95% Ethanol. The gel was left to
polymerize for 25 minutes at room temperature. Thereafter, the ethanol was decanted and the
stacking gel mixture was prepared by mixing 5uL of food colouring, 25uL of 10% APS, 5uL of
TEMED to 2.5mL of Stacking Solution and added to the glass cassette. It was also left to
polymerize for 25 minutes and stored after at 4C.

Casting Gel Electrophoreisis for Prepared Samples

A 1x SDS-Page Running buffer was made from mixing 75mL of the 10x stock to 675mL of
distilled water. To sample 1 (GSTE-N1), 20uL of distilled water, 4uL of 6xPLB and 24uL of Gel
Loading Volume was added. To sample 2 (USP7 Lys) and sample 3 (GSTE Lys), 2uL of 6xPLB
was added. To samples 4 (GST R), 5 (GSTE R), 6 (USP7 D), 7 (GST PD-UP), 8 (GSTE PD-UP),
9 (GST PD-W), 10 (GSTE PD-W), 11 (GST PD-Elt), 12 (GSTE PD-Elt), 4uL of 6x PLB was
added. The protein markers were collected; 42uL of Pre-stained Broad Range Protein Marker and
12uL of BLUeye. These samples were incubated for 10 minutes at 95C. The glass cassette was
removed from storage, the combs were taken out and the gel was submerged in 1x SDS-PAGE
Running Buffer. After the incubation, samples were centrifuged and added to the wells. The gel
was run at 200V for 45 minutes. Once finished, the gels were rinsed with distilled water twice on
the shaker for 5 minutes and submerged with Instant Blue Staining Solution on the shaker for 20
minutes.

Results
Discussion
1. Cover page proper titles (4 marks)

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Methods and materials

Experimental procedure in full sentence
SDS page concentrations, marker/ladder info
Type of gel
Must be into figure captions
6 marks
Results
Figure 1: Bradford standard curve for USP7 concentration
Figure 2: purification gel 1 page (title and caption)
Figure 3: pulldown gel 1 page
Figure 4: protein migration std curve
Discussion (3 pages) (8 marks)
References (2)