Isolation of Protein by Ammonium Sulphate Precipitation

Isolation of Protein by Ammonium Sulphate over-confidenceHypothesisRubisco is a negatively charged protein that weighs 55,000 kDa and is also really soluble. When we add ammonium sulfate to r from each wiz a saturation of 50%, Rubisco can be dislocated utilise ion exchange chromatography and protein electrophoresis.Materials and MethodsIsolation of Protein by Ammonium Sulfate PrecipitationApproximately 300g of fresh spinach leaves were de-ribbed and dried, and then homogenized for 1 narrow in 200mL of cowcatcher. From the homogenized solution, some 50ml was placed into a beaker, which was stirred on a stir plate while 10.90g of solid ammonium sulfate was s let loosely added to reach 37% saturation. The solution was stirred for an additional 10 minutes and then centrifuged at 9,000-xg for 15 minutes. The blastoff ( blastoff I) was resuspended in 4mL of pissing and transferred into a dialysis bag for dialysis against distilled water supply. The supernatant was poured into a beaker and stirred on a stir plate while about 3g of ammonium sulfate was s first-class honours degreely added to reach 50% saturation. After about 15 minutes of stirring, the supernatant was centrifuged for 15 minutes at 7,000-xg. Fol outseting centrifugation, the pellet ( stab II) was resuspended in 4mL of water and transferred into a dialysis bag for dialysis.Ion Exchange Column ChromatographyThe column was equilibrated by speed 30mL of Buffer A (10mM Tris pH 8.0, 3mM EDTA) through. Afterwards, a centrifuge was used to pellet down any solid precipitate out of the dialyzed examples. 1mL of each try out ( gibe I and II) was transferred into labeled Eppendorf tubes and frozen for later use in the SDS-PAGE. Pellet I was diluted a 100-fold and about 4mL of diluted Pellet I and undiluted Pellet II were loaded into separate columns and the samples were allowed to flow through. The flow-through was discarded.10mL of the low common table saltiness buffer (Buffer A + 50mM NaCl) was loaded onto the column and fractions of approximately 2mL were store in separate cuvettes and labeled in order. After blanking the spectrophotometer at 280nm with low salt buffer, the OD renderings of each fraction was metric. This process was repeated using the medium salt buffer (Buffer A + 200mM NaCl) and high salt buffer (Buffer A + 500mM NaCl). The spectrophotometer was blanked with each buffer before the readings of its corresponding fractions were obtained. The fractions with the highest OD reading at 280nm was collected in an Eppendorf tube, labeled, and placed on ice. The column was then washed with 10mL resin cleaning buffer which was discarded in a wash beaker.Protein Electrophoresis30uL of 3X smack Buffer (bromophenol blue, glycerol, dithiothreitol, and SDS) was added to 60ul of each of the 9 samples ( mensuration, homogenate, Pellet I, Pellet I low, medium and high salt buffers, Pellet II low, medium and high salt buffers). The 9 tubes were heated in a water bath fo r 4 minutes. Into a prepared buffer chamber, approximately 20uL of each sample were loaded into separate wells using thin-barrel pipette tips. The mousse was run at 180 Volts for approximately 50 minutes. The gel was removed from the glass plate sandwich and stained in espial/Fixing source for 30 minutes. The gel was destained overnight in Destaining Solution and then dried ona vacuum gel dryer for one hour.Results bow 1 ODwavelength Readings of Protein Fractions for Pellet IOD Reading (280nm)Tube grim sodium chlorideMedium SaltHigh Salt10.202-0.0770.02020.1620.022-0.0833-0.015-0.077-0.05940.2460.003-0.06850.0020.0200.052OD readings (280nm) for the fractions were measured using a spectrophotometer after(prenominal) they eluted from the ion exchange column.Table 2 OD Readings of Protein Fractions for Pellet IIOD Reading (280nm)TubeLow SaltMedium SaltHigh Salt1HI0.121-0.0692HI0.687-0.05931.442-0.017-0.09540.9630.0250.20050.229-0.0490.320OD readings (280nm) for the fractions were measured using a spectrophotometer after they eluted from the ion exchange column.Table 3 Known Protein Standards, Relative Molecular exercising ladings and aloofness Traveled on GelProteinsDistance Traveled (mm)Molecular Weight (Daltons)Phosphorylase b8097,000Albumin13066,000Ovalbumin20045,000 carbonous anhydrase26030,000Trypsin Indicator20,100Lactalbumin14,400The molecular weights of the proteins are known and the distance traveled was gotten by measurement with a ruler from the top of the well to the band of each band.Weight (kDa)Solutions of proteins with known weights were loaded and run in SDS-PAGE and the distance was measured from the top of the well to the bottom of the band.Table 4 titleSample SampleNo. of bandsMigration distance (cm)Molecular Weight (kDa)1Pellet I Low salt2PI Medium salt3PI High salt4PI12.7285Pellet II Low salt12.8266PII Medium salt7PII High salt8Standard12340.81.322.6976645309Homogenate1231.82.32.650.33630The number of bands were tallied, then the m olecular weight was collected as well as the migration distance. The migration was found by measuring from the top of the well to the bottom of the band.Four bands showed in the lane where the standard was loaded instead six, because there were six proteins present in the sample this does not make sense to me. Three bands give sizes appeared in the homogenate and one band in Pellet I and Pellet II low salt.DiscussionRubisco weighs 55,000 kDa according to literature and gel bands that correlate with it that size did not appear in either of the samples. It was predicted Rubisco is very negatively charged and very soluble, therefore it should elute the column at a high salt concentration because a large concentration of salt should be essential to disrupt the bonds created between the negatively charged ion of Rubisco and the positively charged resin, besides the high salt buffer fractions had some of the lowest OD readings which is conflict with our prediction. Instead, the OD read ings were highest with the low salt buffer concentration. This could mean that Rubisco is not as negatively charged and soluble as we predicted. I cannot clearly give over if Rubisco was isolated or not because the gel ripped and a big globe of it was missing due to mishandling. Also, Rubisco might have been lost due to taint because they were no bands that correlated with its molecular weight. GoodThe quality of the experiment could be improved by some additional methods to identify Rubisco since it fixes carbon dioxide. thence they could be a test to show that the protein that was isolated can real fix carbon dioxide.