Purification of Proteins

INTRODUCTION: Protein purification is a series of processes that are followed to isolate a single type of protein from a complex mixture[1] Gel filtration chromatography separates proteins by size [2]. Ion chromatography is that separates ions and polar molecules according to their affinity to the ion exchanger[3]. Affinity chromatography is a separation technique based on molecular conformation [4]. Electrophoresis is the orientation of a charged particle in solution under the influence of an external electric field[5]. Isoelectric focusing is a high-resolution technique in which proteins are separated by isoelectric points in a continuous pH gradient[6]. Hemoglobin is a protein that carries oxygen from the respiratory organ to the tissues and carbon dioxide and proton from the tissues to the respiratory organ[7]. EDTA is a substance that usually used to prevent decalcification and blood clotting[8]. Toluene dissolves the lipid two layers of the cell membrane, breaking down the cell membrane[9].
MATERIALS: Blood sample, 0.9% NaCl, EDTA,  Distilled water, Toluene, 96-well microplates, Eppendorf tubes, pipettes, tubes, pipette tips and centrifugatio
METHOD: Blood samples with EDTA are taken. We thoroughly mixed our blood sample with EDTA. Our sample centrifuged for 5 min at 10000 rpm. Again our sample centrifuged for 5 min at 14800 rpm. Upper plasma portion is discarded with a pipette. salt is added to the sample and shaken gently. Then, centrifugation for 5 min at 3000 rpm. Discard the supernatant. We did this three times. After centrifugation, distilled water as 1.5 times of the underlying layer of red blood cells (~ 750 μl) and toluene as half volume of red blood cells (~ 250 μl) are added. We shaked with hands for 8 min. afterward we centrifuged for 20 min at 3000 rpm. The supernatant is taken carefully with micropipette. Finally we stored our sample at +4 degrees.figüre a) This figure shows us what is blood components.

Figüre b) The image of the blood sample after the first centrifuge. The lines in the photo are white blood cells, red blood cells, and platelets. Image of a mixture of toluene andblood sample after centrifugation. Organelles and cell membrane are found in the supernatant. on the plasma side, it is a mixture of toluene and protein.
DISCUSSION: Our experiment was successful. We isolated proteins from hemoglobin in our blood sample. When we look at the protein we obtained in pcr, we will see two lines. this is because we have two different proteins due to its alpha and beta structure. we have to be sensitive because we isolate something specific.
 if Our lines were not obvious when we first centrifuged. So once again we centrifuge . after this centrifugation there are supernatant and plasma parts. in this centrifugation we obtained our red blood cells, white blood cells and platelets, and we removed the hormones, vitamins and ions present in the plasma part. We split my cells with toluene and we obtained our proteins by centrifugation. we removed the organelles, the lipid layer.
REFERENCES:
1.     https://tr.wikipedia.org/wiki/Protein_safla%C5%9Ft%C4%B1rmas%C4%B1
2.     sigmaaldrich.com/life-science/proteomics/protein-chromatography/gel-filtration-chromatography.html
3.     http://sozluk.ingilizce.tk/?word=ion%20exchange%20chromatography
4.     https://www.thermofisher.com/tr/en/home/references/protein-analysis-guide/affinity-chromatography/what-is-protein-purification.html
5.     https://tr.wikipedia.org/wiki/Elektroforez
6.     https://www.sciencedirect.com/topics/medicine-and-dentistry/isoelectric-focusing
7.     https://tr.wikipedia.org/wiki/Hemoglobin
8.     https://www.kursunkalem.com/biyoloji-terimi/etilendiamin-tetra-asetik-asit-edta/
9.     http://www.journalagent.com/adlitip/pdfs/ADLITIP_23_1_33_43.pdf