University of California, Riverside BCH 162 _Experiment 4: Purification of recombinant dLDH from E.

Experiment 4: Purification of recombinant dLDH from E. coli by metal-ion affinity chromatography Name: Lab Dates: 2/1-2/15 Lab Partner: Lab Report Due Date: 2/22/18

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Objectives Purification The goal of purification is to isolate the specific enzyme lactate dehydrogenase in chicken breast muscle through tissue homogenization, ammonium sulfate precipitation, and pseudo affinity chromatography.

Bradford Assay The purpose of Bradford Assay is to help determine the amount of concentration of protein in each of the fractions taken from the experiment.

Procedures Homogenization in presence of PMSF and 2-ME The homogenization process starts with the addition of PMSF and 2-ME to chicken tissue which will then be mixed in a blender. The homogenate will be transferred to centrifuge tubes, centrifuged for 20 minutes, and filtered through cheesecloth to collect the supernatant.

AmSO4 Precipitation Ammonium sulfate will be slowly added to the supernatant at a ratio of 0.24g for each 1ml of supernatant while mixing the two in a beaker while in the cold room. This solution will be transferred to tubes and centrifuged for 20 minutes giving the 40% cut.

Ammonium sulfate will be added again to the 40% cut at a ratio of 0.13g for each 1 ml

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of 40% cut, mixed and transferred to the centrifuged for 20 minutes which will give the 60% cut.

Resuspension and Desalting The ammonium sulfate pellet will be resuspended with TRIS-ME buffer and kept on ice before transferred to a Sephadex G-25 column. After transferring the resuspended sample to the column, the sample will be desalted with TRIS-ME buffer and collected.

Cibacron Blue Chromatography The desalted sample will be loaded into the Sephadex G-25 column and first washed with TRIS-ME buffer and the elution will be collected and have an absorbance reading. The elution will go through a second wash which contains NAD + and the elution will be collected and have an absorbance reading. The elution will go through a third wash with TRIS-ME buffer and the elution will be collected and have an absorbance reading. The elution will go through the last wash with NADH buffer and have fractions collected along with absorbance readings.

Standard Curve The standard curve will be made by increasing amounts of BSA and decreasing amounts of 10mM Tris-HCl, pH 8.6 being added and mixed into Eppendorf tubes. The first tube will contain no BSA and all 10mM Tris-HCl, pH 8.6 and following tubes will have the trend of increasing BSA and decreasing 10mM Tris-HCl, pH 8.6. After all sample are made 200 microliters of Bradford reagent will be added and the solution

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vortex. Bradford reagent will be added at one-minute intervals until all tubes have reagent. After 15 minutes since the addition of Bradford reagent to the first tube, absorbance readings will be taken using a spectrophotometer.

Dilutions The chicken LDH will be diluted with 10mM Tris-HCl, pH 8.6 to a final volume of 1.0ml or 50 microliters for Crude/Desalted and NADH respectively. The dilutions follow as: crude and desalted being 100-fold dilutions, NADH1,2 and 3 have undiluted and 10fold diluted samples, and an undiluted NADH4. After these dilutions are made, 10 or 50 microliters of these dilutions will be added with 10mM Tris-HCl pH 8.6 and Bradford reagent to obtain absorbance values at wavelength 595. Bradford would be added once all dilutions are made at a 1-minute interval, after 15 minutes have passed since the first addition, absorbances will be taken at a 1-minute interval.

Results Purification During the purification steps for chicken LDH, the volume of the supernatant for the homogenization was 37ml and the pellet was a pinkish color. The homogenization was purified with 8.88 grams of ammonium sulfate and centrifuged giving the 40% cut with supernatant volume of 38ml and a whitish pellet. The 40% cut was treated with 4.94 grams of ammonium sulfate and centrifuged giving the 60% cut.

The graph that plotted A280, protein concentration vs fraction # for chicken LDH is on the carbon copy labeled A280, protein concentration vs fraction # cLDH.

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BSA stock and Tris-HCl Standard Curve Assay #

Protein

200

Sample

10mM

Brandford

added

microgram/m

type

tris-HCl,

Reagent

(microgram

l BSA stock

pH 8.6

(microliter

A595

) (microliter) ) 1 0 0 Blank 800 200 0 2 2 10 BSA 790 200 0.1712 3 4 20 BSA 780 200 0.3307 4 6 30 BSA 770 200 0.5029 5 8 40 BSA 760 200 0.6213 6 10 50 BSA 750 200 0.7368 Micro litter of BSA stock= [(x protein added) / (200microgram/ml)] x 1000 microliters/ mL (2/200 mL) (1000micro liters/mL) = 10 microliters BSA Stock 10mM Tris-HCl= 800 – x microliters BSA stock 800-10= 790 microliters 10mM tris-HCl ph 8.6

X(concentration)= (y(absorbance)-0.0219)/.0744

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Standard Curve 0.8

0.74 f(x) = 74.38 x + 0.02

0.7

0.62

Absorbance A595

0.6 0.5 0.5 0.4

0.33

0.3 0.17

0.2 0.1 0 0

0

0

0

0.01

0.01

0.01

0.01

Concentration mg/mL

Dilutions for fractions of cLDH Used for Table

Sample

Volume

(µl)

5 Assay #

(diluted to)

of sample

Volume of

(µl)

10 mM TrisHCl, pH 8.6 (µl)

1-2 3-4 5 6 7 8 9 10 11

Crude (1:100) Desalted (1:100) NADH1 (undiluted) NADH1 (1:10) NADH2 (undiluted) NADH2 (1:10) NADH3 (undiluted) NADH3 (1:10) NADH4 (undiluted)

10 10 25 5 25 5 25 5 25

Protein determination for cLDH

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990 990 25 45 25 45 25 45 25

Assay #

1 2 3 4 5

Sample

Volume

10 mM

Bradford

Final

A595

Measure

type

to add

Tris-

Reagent

Volume

d

(diluted

(µl)

HCl,

(µl)

(mL)

Protein

to)

pH 8.6

(µg)

Crude Crude Desalted Desalted NADH1

20 60 35 25 10

(µl) 785 740 765 775 790

200 200 200 200 200

1.0 1.0 1.0 1.0 1.0

.2279 .6195 .7433 .6585 .1801

2.7688 8.032 9.69623 8.55645 2.1263

(undiluted 6

) NADH1

10

790

200

1.0

.0771

.7419

7

(1:10) NADH2

100

700

200

1.0

.0795

.7742

(undiluted 8

) NADH2

100

700

200

1.0

.0489

.3629

9

(1:10) NADH3

100

700

200

1.0

.0578

.4825

10

) NADH3

100

700

200

1.0

.0529

.4167

11

(1:10) NADH4

100

700

200

1.0

.0513

.3952

(undiluted

(undiluted )

cLDH Purification Table

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Crude Extract Desalted

Fraction Volume

Concentration of

Total Protein (mg)

(mL) 38.5 4

Protein (mg/mL) 13.84 28.12

532.84 112.48

5

0.077

0.385

Ammonium sulfate fraction “peak” NADH fraction from Cibacron Blue column Total protein= (Fraction volume) x (Concentration of Protein) Crude Extract= 38.5 mL(13.84mg/mL) = 532.84 mg Desalted Ammonium sulfate fraction= 4 mL (28.12 mg/mL) = 112.48 mg “peak” NADH fraction from Cibacron Blue column= 5 mL mg/mL) = 0.385 mg Discussion During the homogenization step a total of 37mL of supernatant was collected and after adding ammonium sulfate and centrifuging the solution, a total of 38mL of supernatant was collected from the 40% cut. During this purification the supernatant increased very minimally which says that the 40% cut had lost some protein to the pellet after centrifuge. Because the addition of ammonium sulfate would increase the volume of supernatant by stabilizing the proteins and not allowing them to denature it safe to say that some protein was lost to the pellet during centrifuge. While doing the pseudo-affinity chromatography and collecting the fractions, a peak absorbance was identified at fraction 2. The peak in absorbance at this fraction

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indicates that majority of the protein was eluted with NADH into this fraction compared to the others. Since all the following fractions after the second were decreasing it solidifies that the most amount of protein were in fraction 2. For determination of protein concentration for chicken LDH, the original dilutions gave absorbance values that were too low for the standard curve. A new set of dilutions had to be made were higher amounts of dilutions and less amount of buffer had to be added. The absorbances from the first set of absorbance had low values which means that there wasn’t enough concentration in the cuvette when the reading was taken. After changing the dilutions making the samples less diluted the absorbance values increased and were in range of the standard curve. Looking at the amount diluted sample and buffer added, the absorbance values followed a trend were more of the sample was added the higher the absorbance and vice-versa. The trend of sample added to absorbance value shows that there wasn’t anything weird happening in the samples.

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