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Michelle Grau Lactate Dehydrogenase Protein Purification and Analysis Laboratory September 17 2009-October 16 2009- Notebook Pages

: 58-96

Introduction: In this lab investigation, the aim was to purify and analyze Lactate Dehydrogenase (LDH) enzyme from chicken breast. Protein purification is essential when studying the function, structure, and interactions of a particular protein. It’s important to determine the concentration of protein if using it a variety of physical analytic methods such as steady-state kinetics or ligand binding that rely on molar measurements. LDH is an enzyme found in most plants and animals and plays a role in glycolysis for the formation of ATP.1 This enzyme catalyzes the reaction of pyruvate and NADH to form lactate and NAD+, while at the same time it can catalyze the reverse reaction if there’s a large concentration of lactate. If you wanted to purify LDH, or any protein, you must start with a tissue sample containing the protein. Most proteins are typically found within a cell, so the tissue must be subjected to a homogenizing process in order to break cell walls and release protein. If the protein is in solution, you expose it to a selective precipitation. Selective precipitation of proteins can be used as a rough method to recover a desired protein in a purification. This process depends on the physical or chemical interaction between the protein and the precipitating agent. In this lab, (NH4)2SO4 is used to precipitate out LDH. Dialysis is a process of separating molecules in solution by differences in rates of diffusion across a semi-permeable membrane. Dialysis can most often remove a large amount of small impurities in a heterogeneous solution containing your protein. We used dialysis in this lab to remove the excess, unwanted (NH4)2SO4 and other small impurities while simultaneously exchanging the extraction buffer with dialysis buffer. Affinity chromatography is used to obtain a specific substance if it’s mixed in a heterogeneous solution. Columns used for affinity chromatography are typically composed if inert, chemically stable polymers, that have specific binding proteins or molecules. You would use a column that was composed of a specific molecule to which the protein of interest would bind to with a high affinity. Once the protein solution is applied to the column, all substances and proteins that do not bind or that bind loosely to the column are removed with buffer washes. Then the column is washed with a solution to which the desired protein binds strongly to and is isolated. In this lab we used a Cibacron blue affinity column to purify LDH. This molecule mimics the shape and charge characteristics of pyridine nucleotides to which dehydrogenase proteins frequently bind to. To obtain pure LDH from the column, it was washed with an NADH solution because of the high affinity LDH has for NADH. Once your protein is purified, there are many techniques to determine the purity and concentration of your protein. First you typically run an activity assay if the protein has enzymatic properties, to determine which fractions from the chromatography contain the protein. SDS-PAGE gel electrophoresis is a good method to use for determining the purity of the protein. This method separates proteins according their molecular weight and length of polypeptide chain. Proteins all exhibit the same charge per unit mass due to the binding of SDS resulting in fractionation by size and mass. Using the gel from SDS-PAGE, you can detect the protein using a specific and visible antibody against the protein. Bradford Assays are a routine method for determining protein concentration. A standard curve made with known protein concentrations is constructed using Coomassie blue dye which binds to all proteins and absorbs light at 595nm. Based on the absorbance of the solution containing the protein of interest, using the standard curve you can determine a relatively accurate protein concentration. The Edelhoch method measures denatured protein concentration, based on the absorbance at 280nm and the extinction coefficient that is determined by the number of tyrosines, tryptophans, and cysteins in the polypeptide chain.2 All these methods described were used in the lab to determine the purity and concentration of LDH protein.

Aims of Experiment: The purpose of this experiment was to extract and purify LDH enzyme from chicken breast muscle using a variety of techniques including centrifugation, selective protein precipitation, dialysis and affinity chromatography. Many different analytical methods were employed to determine the presence, purity and concentration of LDH such as activity assays, SDS-PAGE, Western Blot, Bradford Assay, Edelhoch, and QAAA. Results: Figure 1. Flowchart Depiction of LDH Protein Purification Procedure and Analysis
Step 1: 50g of Chicken Breast in 75mL of Extraction Buffer Extraction Buffer: 10mM TrisHCl pH 8.6, 1mL 2mercaptoethanol, 100mM PMSF, 1mM ethylene diamine Chicken tissue and extraction buffer homogenized using 4x 30sec bursts allowing 10 seconds between bursts Step 2: Centrifugation 27,000 x g, 4°C for 20 min in 250mL conical vials Supernatant Collected – 52mL (Crude Homogenate) Step 3: Ammonium Sulfate Used 20.28g (NH4)2SO4 (0.39g of (NH4)2SO4 per mL of supernatant) Added in cold room slowly over 15 minutes and then stirred for an additional 15 minutes. Step 4: Centrifugation Same conditions as above. Supernatant Collected- 52mL (Ammonium Sulfate Supernatant) Step 5: Resuspended Pellet in 5mL of Extraction Buffer Same buffer as described above. Suspension contained protein and (NH4)2SO4 Volume of pellet in extraction buffer- 7.2mL Step 6: Dialysis Dialyzed suspension two times in 1L of dialysis buffer Dialysis Buffer: 10mM TrisHCl pH 8.6, 5mM 2-mercaptoethanol Saved 3 aliquots of dialyzed sample Step 7: Affinity Chromatography

Used a Cibacron Blue Affinity Column Absorbance of all fractions was measured with a UV-Vis Spectrophotometer at 280nm. Blank was measured with milli-Q water. Absorbance of PMSF buffer was about 0.004. Absorbance of each fraction was below 0.1 Abs before moving on to each consecutive wash. Flow Through Tris PMSF Wash 10mL of NAD+ wash: Tris PMSF 10mM TrisHCl pH 8.6, #1: Wash #2:
Same solution as wash #1

10mL of NADH wash:
10mM TrisHCl pH 8.6, 0.5mM 2-mercapto ethanol, 1mM NADH

0.5mM 2-mercapto ethanol, 10mM TrisHCl pH 1mM Lithium Lactate, 8.6, 0.5mM 2+ mercaptoethanol, 1mM 1mM NAD PMSF

Tris PMSF Wash #3:
Same solution as wash #1

Fractions: 1-3

Fractions: 4-9

Fractions: 11,12

Fractions: 13-16

Fractions: 17,18

Fractions 19,20

Step 8: Analysis of LDH Purification

Activity Assays of LDH Samples Analysis parameters located in Table 1

SDS-PAGE Gel Electrophoresis Analysis parameters located in Figure 2

Bradford Assay for determination of LDH concentration

Edelhoch Analysis Analysis parameters located in Table 2 and Figure 5

Analysis parameters located in Table 1 Western Blot analysis Analysis parameters located in Figure 4 LDH mass determination via BenchMarkTM Protein Ladder Standard Curve Analysis parameters located in Figure 3 Bradford Assay for determination of LDH concentration in pooled fractions Analysis parameters located in Table 2

A crude homogenate of chicken breast was obtained and to this, ammonium sulfate was added to precipitate LDH. This precipitate was treated as described in the flowchart in Figure 1. Following dialysis, the LDH sample was subjected to a Cibacron blue affinity column. LDH activity assays were performed to determine which samples and fractions contained a significant amount of LDH. The results of the LDH activity assays are located in Table 1. The highest enzyme activity was observed in the crude homogenate. Fraction 17 produced the highest observed activity from the chromatography fractions. A higher enzyme activity was observed in the (NH4)2SO4 supernatant than the (NH4)2SO4 dialyzed protein solution. Bradford Assay Analysis: Table 1: LDH Concentration, Purity, and Yield Determination via Bradford Assay
Sample Dilution Abs LDH Activity
(μmol NADH min1 mL-1)1

Crude Homogenate 12,500 0.141 75.19 0.0028

(NH4)2SO4 Supernatant 12,500 0.065 13.11 0.0003

(NH4)2SO4 Dialyzed 125 0.45 18.95 0.0130

Fraction #11 125 0.347 33.99 0.0096

Fraction #12 125 0.359 38.57 0.0100

Fraction #17 125 0.613 45.86 0.0184

Fraction #18 125 0.396 8.51 0.0112

Fraction #17 + #186 200 0.221 27.19 0.0066

Diluted Protein Concentration
(mg/mL)

Undiluted Protein Concentration
(mg/mL)

34.810 52.0 1810.099 3909.88 2.160 1.00 100.00

3.353 52.0 174.338 681.72 3.910 1.81 17.44

1.627 7.2 11.715 136.44 11.647 5.39 3.49

1.201 5.0 6.004 169.95 28.307 13.11 4.35

1.250 5.5 6.877 212.135 30.846 14.28 5.43

2.302 6.0 13.810 275.16 19.924 9.22 7.04

1.404 6.0 8.421 51.06 6.063 2.81 1.31

1.362 12.5 17.025 339.875 19.963 9.24 8.69

Volume (mL)2 Total Protein
(mg)

Total LDH Activity (μmol
NADH min -1)

Specific LDH Activity (μmol
NADH min -1 mg-1)3

Fold Purification4 Yield (%)5

Table 1: Bradford Assay standard curve was constructed using IgG protein concentrations between 1.25 μg/mL-25.0μg/mL. BioRad Assay Reagent was added to standards, samples, and fractions that were diluted in 50mM KH2PO4 buffer at pH 8.0. The final volume was 1mL. The blank was measured with 50mM KH2PO4 buffer at pH 8.0. Absorbance was measured at 595nm using a UV-Vis Spectrophotometer. Path length was 1cm. The equation of the standard curve determined through the Bradford Assay was: y = 0.0302x + 0.056. Dilutions were made for samples to obtain an absorbance between 0-1. 1LDH ActivitiesActivity of 10μL LDH was determined via time course measurements using the UV-Vis Spectrophotometer, monitoring the NADH production by following the absorbance at 340nm. Duplicates were measured for samples with significant enzyme activity. 2 Volumes- See flowchart. 3 Specific LDH Activity = LDH Activity/ Undiluted LDH Concentration. 4 Fold Purification = Specific LDH Activity/ Crude Homogenate Specific LDH Activity. 5 % Yield = (Total LDH Activity/ Crude Homogenate LDH Activity) *100. 6 The absorbance and both diluted and undiluted concentrations for this fraction was determined by the Bradford Assay as described in Table 2. Absorbance value is an average of two measurements. The enzyme activity was estimated based on the average of the two fractions. All the subsequent calculations are therefore also estimations.

A Bradford analysis was used to determine the concentration of LDH in a selection of samples and fractions that had a significant observed enzyme activity. Specific parameters for the construction of the Bradford Assay standard curve and the results of this analysis are located in Table 1. The total mass of protein was determined based on the measured volume of each sample or fraction and its corresponding protein concentration. Total LDH activity was also calculated based on the total volume of each sample or fraction. The specific LDH activity, fold purification, and % yield were calculated as described in Table 1. The crude homogenate had the

largest observed amount of LDH as well as the greatest total LDH activity and % yield while producing the lowest specific LDH activity and fold purification. Of the column fractions (not combined), 17 had the largest observed LDH concentration, mass and total LDH activity, while 12 had the largest specific LDH activity, fold purification and % yield. The (NH4)2SO4 supernatant had a larger protein concentration, mass, total LDH activity, and % yield as compared to the (NH4)2SO4 dialyzed protein solution which had a larger specific LDH activity and fold purification. After the SDS-PAGE analysis (Figure 2), fractions 17 and 18 were combined because they had the largest observed specific LDH activity of the fractions that resulted from the NADH wash. An enzyme activity was estimated based on the average activity of both fractions. The absorbance and concentration was determined through a different Bradford Assay as described in Table 2. The same calculations were made for the combined fractions that were made for all the samples and individual fractions. This fraction had the largest mass, total LDH activity and % yield than all the other fractions. The overall yield of the pooled fractions was 17.025mg LDH/50g chicken breast. SDS-PAGE Gel Analysis: An SDS-PAGE gel was run to determine the purity of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 LDH in select samples and fractions that had observed enzyme activity. An image of the gel is in Figure 2. An LDH control was used as a reference for determining which band corresponded to LDH. The lanes with crude homogenate, dialyzed (NH4)2SO4 protein, F11, F12, F13, F17, and F18 all appeared to have a band that corresponds to the LDH control band. Fractions 11, 12, Figure 2: SDS-PAGE-Each sample was made in SDS-sample buffer: 0.0583M TrisHCl, 5% (v/v) and 17 have the thickest glycerol, 1.713% (w/v) SDS, 10mM DTT, 0.0017% (w/v) Bromophenol blue. 15μL of each sample and fraction was added to each well. 5μL of BenchMark Protein standard was added to wells 1 an 14, and bands. There were a lot of 2μL was added to well 15. Lane 1: Standard, Lane 2: Crude Homogenate, Lane 3: Na2SO4 observed bands in the crude Supernatant, Lane 4: Na2SO4 Dialyzed Protein, Lane 5: Fraction (F)11, Lane 6: F12, Lane 7: F13, Lane 8 F14, Lane 9: F17, Lane 10: F18, Lane 11: F19, Lane 12: F20, Lane 13: Control LDH protein, homogenate lane that didn’t Lane 14: Standard, Lane 15: Standard. SDS-PAGE was run in running buffer: 0.025M TrisHCl, correspond to LDH. In all the 0.192M glycine, 0.1% (w/v) SDS. 12% acrylamide gel was used. Proteins were visualized with Coomassie blue staining. fractions, there were other bands that didn’t correspond to LDH, however, there were a significantly less number of bands in the fractions as compared to the crude homogenate as well as the (NH4)2SO4 supernatant and (NH4)2SO4 dialyzed protein. The SDS-PAGE results were also analyzed for the determination of the molar mass of LDH. Comparison of the experimental molar mass to the actual molar mass was used for confirmation that the protein was LDH. Using the largest molecular weight protein band as a baseline, a standard curve was plotted, located in Figure 3. The bands that corresponded to LDH in each of the lanes were determined via the Western Blot analysis (Figure 4). In addition, the LDH band in lane each was predicted based on the corresponding control lane containing LDH.
TM

Figure 2. SDS-PAGE Gel of LDH Samples and Fractions

15 14 13 12

Figure 3. Standard Curve of BenchMarkTM Protein Ladder from The distance was measured SDS-PAGE Gel For LDH Mass Determination between the LDH band in lane 17 was measured to and the baseline. This distance, 3.05cm, was used in the equation of the standard curve to calculate for an experimental molecular weight of LDH. The distance measured was 3.05cm. The experimental molecular mass was determined to be 36,791g/mol. The percent difference was <1% between the experimental mass measurement Figure 3: Using the SDS-PAGE gel, the distance of each protein benchmark was measured and the actual molecular mass from the baseline. The baseline was the largest molecular weight benchmark. which is 36514.4g/mol.

Western Blot Analysis: Using the second SDSPAGE gel, a Western Blot analysis was accomplished to confirm the presence of LDH in each sample and fraction. A 1° and 2° antibody was used for detection of the LDH protein and for visualization purposes. A visual result of the Western Blot is located in Figure 3. Antibody treatment resulted in distinguishable bands on the membrane, suggesting the presence of LDH. Figure 4. Results of Western Blot Analysis Using SDS-PAGE gel

Dialyzed (NH4)2SO4

F17

F18

LDH Control

Crude Homogenate

F11

F12

Figure 4: SDS-PAGE gel was used for the Western Blot analysis. Gel was transferred to a
nitrocellulose membrane. Membrane was probed with 1° antibody (goat-Anti-rabbit-LDH-antibody) was used for specific LDH detection on the membrane. Membrane was then probed with a 2° antibody (rabbitAnti-goat-IgG-alkine-phosphitase-conjugate) was used for LDH visualization. Spots from left to right: crude homogenate, dialyzed ammonium sulfate, F11, F12, F17, F18, LDH control. This figure was enhanced to better visualize the presence of LDH.

Accurate LDH Concentration Determination via Edelhoch and Bradford Assay Analysis: Fractions 17 and 18 were combined and dialyzed against 2 volumes of dialysis buffer Lactate Dehydrogenase Carbonic Anhydrase (10mM TrisCl pH 8.6, Concentration Concentration 0.5mM 2mercaptoethanol) and this Edelhoch Analysis -0.69mg/mL LDH sample was subject to a subsequent Bradford Bradford Assay Analysis 1.3618mg/mL 1.3236mg/mL Assay and an Edelhoch analysis. A sample of Quantitative Amino -0.691mg/mL Carbonic Anhydrase Acid Analysis (CA) was also analyzed Table 2: Bradford Assay Analysis: A Bradford Assay standard curve was determined using the parameters as in both these techniques described in Table 1. The concentration of LDH and Carbonic Anhydrase (CA) was determined using the for comparison purposes equation of the standard curve: (y = 0.0288x + 0.0299) The absorbance of LDH and CA was 0.226 and 0.2205 respectively. Edelhoch Analysis: The absorbance of LDH and CA samples were obtained in 50mM KPO buffer and to probe the accuracy pH 7.4 with and without 6M GdnHCl measured at 280nm using a UV-Vis Spectrophotometer. The final volume and limitations of various was 1mL. The blank was measured with dialysis buffer. The value of ε was determined for LDH and CA using the Pace et al. reference . ε = 40,535M cm for LDHA and ε = 43,105M cm for CA. Abs techniques used for (CA-denat.)= 0.2112131, Abs (CA-nat.)=0.311365. Beer’s Law was used to determine the concentration of CA. determining protein Quantitative Amino Acid Analysis: QAAA was completed on the CA sample to give an accurate concentration measurement. concentration. For the Edelhoch analysis, absorbance values were measured at 280nm for native and denatured samples of LDH and CA. 6M Guanidine hydrochloride (GdnHCl) was used to denature the proteins. An extinction coefficient was determined for both LDH and CA denatured in GdnHCl using the Pace et. al. reference.2 Using the extinction coefficient for CAdenatured (located in Table 2), a concentration for CAdenatured was determined and the results are located in Table 2. The values of Absdenatured and Absnative were measured for CA at 280nm, and knowing the value of εdenatured, the
Table 2. Accurate Concentrations of Pooled LDH Fractions and Carbonic Anhydrase Protein Determined via Bradford Assay and Edelhoch Analysis
4 Denatured 2 Denatured -1 -1 Denatured -1 -1

value of εnative at 280nm was calculated for CA.

Figure 5. Absorbance Scan of Natured LDH Sample The LDH protein concentration could not be determined using the Edelhoch analysis because a large absorbance peak at 260nm completely covered the peak at 280nm. A broader absorbance scan was obtained for the natured LDH and is located in Figure 5. Absorbance peaks at 260nm and 340nm suggested the presence of NADH in the sample. The absorbance at 340nm for the native LDH was 0.5020482. Based on
2.5 2.0 1.5 Abs 1.0 0.5 0.0

Figure 5: This absorbance scan was of the combined Fraction 17 and 18 sample in dialysis buffer: 10mM TrisCl pH 8.6, 0.5mM 2-mercaptoethanol, diluted in 50mM KPO4 buffer pH 7.4 . This was one of the Edelhoch analysis samples. The parameters of the Edelhoch analysis are located in Table 2. ε for NADH = 6220M-1cm-1.
340

the extinction coefficient for NADH at 340nm, the concentration of NADH was determined to be 0.2675mg/mL

LDH and CA protein concentrations were determined using a Bradford Assay. The standard curve equation and protein concentrations are located in Table 2. The results of the Bradford Assay for the combined LDH samples are also located in Table 1. The sample of CA protein was sent to Texas A&M University Protein Chemistry Laboratory for quantitative amino acid analysis (QAAA) and the determined concentration is incorporated into Table 2 as well. It was observed that the Edelhoch method and QAAA provided a very similar value for the concentration of CA protein. The Bradford Assay concentration for CA was significantly larger than the other two methods. The only concentration determined for the combined LDH samples was from the Bradford Assay analysis. Discussion: The results of the first Bradford Assay (Table 1), provide concentration data of LDH in each sample and fraction while also providing information about the purity of LDH. The specific LDH activity and fold purification are measures of purity; the samples with larger values of specific LDH activity and fold purification are more pure than samples with lower values. The chromatography sample with the largest LDH concentration was fraction 17, and the sample with the highest purity was fraction 12 based on this analytical technique. The results of the (NH4)2SO4 dialyzed sample in Table 1 provided contradictory information. The total mass of LDH for this sample was less than the combined mass of LDH from all the fractions. This wasn’t possible because all the LDH in the fractions came from (NH4)2SO4 dialyzed sample. It is possible that an alternate dilution was measured and not correctly accounted for when calculating LDH enzyme activity from the activity assays. The SDS-PAGE results provided conclusive evidence to evaluate the presence and purity of LDH in all of the samples and fractions. From the gel in Figure 2, LDH is present in the crude homogenate, dialyzed (NH4)2SO4, Fractions 11, 12, 13, 17, 18, and possibly in the (NH4)2SO4 supernatant. These samples and fractions all had a band that corresponded to the same distance of the band in the LDH control lane. Further evidence that these were all LDH bands was provided through the Western Blot analysis, however it was difficult to visualize the bands for the (NH4)2SO4 supernatant and fraction 13. From the control LDH lane it was determined that LDH protein is a single species or produces only one band. This means that LDH is either a monomer, or an oligomer composed of only one type of monomer. It seems that fractions 11,12, and 17 contain the most LDH protein based on the thickness of the band from the SDS-PAGE gel. If I had a chance to change which fractions were combined, I would probably use fractions 11, 12, and 17 due the high LDH concentration, as well as the fact that they all had the same level of purity. I assume that the only difference between the proteins that came out with the NAD+ wash as opposed to the NADH wash was the confirmation of the protein. I also think that the LDH that came out with NAD+ is just as pure as the LDH that came out with the NADH. This can be concluded by looking at the different fractions from each wash on the SDS-PAGE gel. The SDS-PAGE gel however, does not provide evidence of non-protein impurities. The presence of impurities in each of the samples was established by the appearance of other bands than the LDH protein. The crude homogenate had the most significant amount of

impurities. The (NH4)2SO4 supernatant and dialyzed (NH4)2SO4 samples both had impurities, but in much less concentration than the crude homogenate. All the fractions from the affinity chromatography had a significantly less amount of impurities as compared to the crude homogenate. It would appear that all the fractions had relatively the same purity of LDH protein as the impurity bands in each fraction that had LDH, appeared to be the same intensity. The impurities are most-likely other proteins found in chicken breast muscle that also have a binding affinity to NADH, NAD+, and/or molecules containing pyridine groups, as they bound themselves to the Cibacron blue column that mimics these molecules. Based on the SDS-PAGE protein BenchMarkTM standard curve located in Figure 3, the estimated molecular mass was calculated to be 36,791g/mol using the equation of the line. This estimate is very close to the known mass of LDH, which is 36,514.4g/mol, as determined by ExPASy Proteomics Service online. The percent difference is <1%. The minor disparities between the estimate and the actual mass could be attributed to the nature of an SDS-PAGE. In this analysis, the protein sample is denatured and non-polar regions become covered with SDS molecules. This adds to the molecular mass of the protein. It is a reasonable explanation as our estimated mass was slightly higher than the actual mass. In an SDS-PAGE, the protein is denatured allowing the nonpolar regions of the SDS molecules to associate with the nonpolar regions of the protein, preventing re-naturation. In this sense, the molecular mass determined by SDS-PAGE is of the proteins primary structure (plus the mass of the SDS molecules associated to the protein). If the protein monomer associates with other monomers in its quaternary structure, you would not be able to determine this when using SDS-PAGE. From the SDS-PAGE gel it appears that there was LDH protein present in the lane containing the ammonium sulfate supernatant, however, this lane is rather skewed probably due to the high concentration of ammonium sulfate. Presence of LDH in this sample provides evidence that not all of the LDH protein in the crude homogenate precipitated. To precipitate more of the LDH protein, more ammonium sulfate should have been added A strange result was observed for the dialyzed ammonium sulfate sample in the SDSPAGE gel (Figure 3). It seems that not very much LDH protein was present, rather, a lot of a protein slightly higher in molecular mass than LDH was present with only a small amount of LDH. This would not seem correct because the dialyzed ammonium sulfate sample should have had a large LDH concentration. It’s possible that a sample was mislabeled and incorrectly loaded into the gel. From the Edelhoch analysis an accurate CA concentration was determined as it was almost the exact concentration established from the QAAA. QAAA is a very precise and reliable method used to determine protein concentrations when the protein is pure. The Edelhoch method could not be used for the LDH protein because it was determined that a large concentration of NADH was present in the sample. Both the natured and denatured LDH samples had large absorbance peaks at 260nm and 340nm, which are two wavelengths for which NADH has a high absorbance. The peak at 260 completely covered the peak at 280 which was supposed to be used to determine the LDH protein concentration with the calculated extinction coefficient for denatured LDH protein in GndHCl at 280nm. The fact that so much NADH was present in solution stresses how tight LDH binds to NADH. To improve the previously described outcome, it is possible that the LDH could be dialyzed with pyruvate such that the NADH would be converted to NAD+, and because LDH has a much lower binding affinity for NAD+, it is possible that the NAD+ could be eliminated through this dialysis. The calculation for CA concentration via a subsequent Bradford Analysis appeared rather large as compared to the concentrations determined through Edelhoch and QAAA. The reason for this could be that the BioRad Protein Assay Reagent (which turns blue upon protein binding) binds to all proteins present in solution. If the LDH sample had other protein impurities, which

was observed in fraction 17 and 18 from the SDS-PAGE gel, there would be a larger production of blue color, increasing the absorbance of the sample. This is a limitation of the Bradford Assay analysis. If this were the case, than the concentration of the LDH calculated from this Bradford Analysis may also be higher than the actual concentration. Because the Edelhoch method didn’t work for LDH and there was no QAAA for LDH, there were no other concentrations of the combined LDH fractions to use for comparison purposes. QAAA is the most accurate method to determine protein concentration, because a concentration of each amino acid is determined in the sample, and based on the number of a particular amino acid present in the protein, an accurate protein concentration can be deduced. The limitation of this method would be if other protein impurities were present in the solution, you would obtain an inaccurate concentration calculation. The Edelhoch method is also very accurate, however, there are a few limitations. For example, if there’s an impurity in the protein solution that affects the absorbance at 280nm, like we saw with LDH containing NADH, you will not be able to use this method. Conclusions: From the results and analysis it can be concluded that LDH was successfully purified from chicken breast muscle. There were still some minor impurities following the affinity chromatography as determined through analysis of the SDS-PAGE, however the protein appeared to be purified by at least 90% from the crude homogenate. The Western Blot analysis confirmed that the protein was LDH. The final concentration of the pooled LDH fractions was 1.362mg/mL, as determined by Bradford Assay, with a 8.69% yield. From the CA study, it was concluded that the Edelhoch method and QAAA are very accurate methods of demining protein concentration, however QAAA will typically be the most accurate if the protein sample is pure.

References: 1. Campbell, N.A. and J.B. Reece. 2005. Biology, 7th edition. Benjamin Cummings, San Francisco. 2. Pace, C. Nick, Felix Vajdos, Lanette Fee, Gerald Grimsley, and Theronica Gray. "How to Measure and Predict the Molar Absorption Coefficient of a Protein." Protein Science 4 (1995): 2411-423. Print.