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EXPERIMENTS 2.1-2.4: CELLFRACTIONATION, PROTEIN ASSAY,        SDH ASSAY AND          SDS-PAGEMichael Truck - Biology 104 -...
PURPOSECell Fractionation - To Understand the laboratorytechnique that uses differential centrifugation toseparate the dif...
EXPECTED RESULTSMitochondrial fractions should have highest SDHspecific activity (electron transport)Homogenate fraction sh...
SUMMARY OF PROCEDURECell Fractionation: homogenized beef liver, added somebuffer, centrifuged at different speeds and inte...
SDH Assay: prepared dilutions of each fraction, addedenzyme mixture and then placed in 37°C water bath;read absorbance at ...
RESULTS – BSA STANDARD CURVETube   Dilution Factor         BSA Volume (mL)         ddw Volume (mL)         [BSA]ug/mL Conc...
RESULTS – CELL FRACTIONSTube    Dilution Factor          Volume (mL) Fr. + d.w.             ABS average           Conc. (m...
RESULTS – SDH ASSAYTube                        t=0                t=3                  t=151                  Blank    -  ...
0                                                 25                                                      50              ...
DISCUSSION Here we can see that the Homogenate fractionshowed the highest enzymatic activity and themitochondrial fraction...
MORE SOURCES OF ERRORthe fractionation process was not carriedout long enough for the cells tofractionate.The absorbance r...
DOES ANY 1   HAVEQUESTIONS     ?
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2.1 2.4 presentation

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2.1 2.4 presentation

  1. 1. EXPERIMENTS 2.1-2.4: CELLFRACTIONATION, PROTEIN ASSAY, SDH ASSAY AND SDS-PAGEMichael Truck - Biology 104 - TA: Elizabeth Braschayko
  2. 2. PURPOSECell Fractionation - To Understand the laboratorytechnique that uses differential centrifugation toseparate the different components of the cell,resulting in nuclear, mitochondrial, microsomal, andsoluble fractions.Protein Assay - To determine the proteinconcentration of each fraction obtained.Succinate Dehydrogenase (SDH) Assay – Used totest the enzymatic activity of SDH in the differentcellular fractions.SDS-PAGE - to identify the unknown proteins inthe mitochondrial proteome
  3. 3. EXPECTED RESULTSMitochondrial fractions should have highest SDHspecific activity (electron transport)Homogenate fraction should have highest proteinconcentration but lower SDH activity thanmitochondria (contains unbroken cells + mixture oforganelles)Soluble fraction should have lowest proteinconcentration and lowest SDH specific activity(smallest cellular particles, lowest amount ofproteins)
  4. 4. SUMMARY OF PROCEDURECell Fractionation: homogenized beef liver, added somebuffer, centrifuged at different speeds and intervals toget the different cellular fractions.Protein Assay: used Bovine Serum Albumin (BSA) to makestandard curve using known BSA concentrations; madedilutions of cell fractions and calculated proteinconcentrations using linear equation of standard curve
  5. 5. SDH Assay: prepared dilutions of each fraction, addedenzyme mixture and then placed in 37°C water bath;read absorbance at t=0 min., t=3 min. and t=15 min.;We then used this data with the protein concentrationsto calculate the specific activity of SDH in each sampleSDS-PAGE: Added acetone to protein sample. Incubatedfor 40 mins., centrifuged, disposed of the supernatant,dried the pellet. Added 8ug buffer and 10ul of 2x gelloading dye and placed in water bath. Then loaded ontoSDS-PAGE gel.
  6. 6. RESULTS – BSA STANDARD CURVETube Dilution Factor BSA Volume (mL) ddw Volume (mL) [BSA]ug/mL Conc. BSA Amount in Assay (ug) Abs. @ 5951 0 1 0 1000 100 0.2562 4/5 0.8 0.2 800 80 0.2523 3/5 0.6 0.4 600 60 0.2414 2/5 0.4 0.6 400 40 0.2355 1/5 0.2 0.8 200 20 0.2206 1/10 0.1 0.9 100 10 0.2027 - - 1.0 0 0 0.08 - - - 0 0 0.079
  7. 7. RESULTS – CELL FRACTIONSTube Dilution Factor Volume (mL) Fr. + d.w. ABS average Conc. (mg/mL)H1 1/10 0.5+4.5 0.234 2.22H2 1/20 0.5+9.5 0.064N1 1/5 0.5+2.0 0.286 3.21N2 1/10 0.5+4.5 0.143M1 1/5 0.5+2.0 0.386 7.02M2 1/10 0.5+4.5 0.241S1 1/5 0.5+2.0 0.342 5.35S2 1/10 0.5+4.5 0.188
  8. 8. RESULTS – SDH ASSAYTube t=0 t=3 t=151 Blank - - -2 H 0.994 0.373 0.2563 N 0.922 0.318 0.1974 M 1:5 0.952 0.383 0.2865 M 1:10 0.910 0.334 0.2306 M 1:25 0.894 0.332 0.2657 S 0.930 0.480 0.298Tube SDH Specific Activity2( Homogenate, 1:2) 1413(Nuclear, 1:2) 1244(Mitochondrial, 1:5) 34.65(Mitochondrial, 1:10) 44.26(Mitochondrial, 1:25) 26.87(Soluble, 1:2) 74.3
  9. 9. 0 25 50 75 100 125 150 2( Homogenate, 1:2) 3(Nuclear, 1:2) 4(Mitochondrial, 1:5) SDH Assay 5(Mitochondrial, 1:10) 6(Mitochondrial, 1:25)SDH SPECIFIC ACTIVITY 7(Soluble, 1:2)
  10. 10. DISCUSSION Here we can see that the Homogenate fractionshowed the highest enzymatic activity and themitochondrial fractions showed the lowestactivity. This also shows that the 1:10 dilution ofmitochondria is higher than the 1:5 dilutionwhich is wrong. This data is all contradictory asto the correct outcome of what the resultsshould be. Some sources of error could be thatthe mitochondria was not fully broken open inthe homogenization step. This being said, SDHcould have not been interacting with the DCPIPdue to the fact that the mitochondrial wall wasnot broken open.
  11. 11. MORE SOURCES OF ERRORthe fractionation process was not carriedout long enough for the cells tofractionate.The absorbance readings contained Errorfrom the spec-20 machine having dirtytubes or from malfunction of the machineitselfHuman error (vague - covers many things)
  12. 12. DOES ANY 1 HAVEQUESTIONS ?

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