Opening the book…
I. Introduction
Gel filtration chromatography is also known as size exclusion, molecular sieve, gel permeation chromatography. The primary objective of gel filtration is to achieve rapid separation of molecules based on size. The gel, which is in bead form for easy column packing, consists of an open, cross-linked three-dimensional molecular network of pores into which molecules of less than the pore size may penetrate. The pores within the beads are of such sizes that some are not accessible to large molecules, but smaller molecules can penetrate all pores. The extent to which a molecule enters the pores depends on its shape as well as molecular weight. Therefore, separation depends on the difference in the ability of various molecules to enter these pores. Proteins above the molecular weight exclusion limit cannot enter the pores. Consequently, they come through the column as a sharp peak in the void volume.
II. Materials and Methods
Materials:
1. Protein sample (crude albumin from Exp. 2A mixed with Vit. B12)
蛋白樣品[實驗2A的粗白蛋白加維他命B12]
2. Buffer: 50 mM Tris-HCl, pH 7.0, 50 mM NaCl
3. Column, test tubes and test tube rack
4. Pre-swollen Sephadex G-50 (fine particle), degassed.
除氣、預先吸水膨脹過的Sephadex G-50膠體過濾凝膠溶液
[請注意凝膠溶液只能以玻棒輕輕攪拌，不可以攪拌子與震盪器劇烈攪拌，會導致膠體顆粒碎裂，影響分離效果。]
5. Spectrophotometer and quartz cuvette (handle with care!!)
石英比色管-價錢昂貴請特別小心
Procedure:
1. Assemble column apparatus. Mark the 50 ml solution level on the column.
將管柱組裝好，利用量筒量取50 ml的水，倒入管柱中，在管柱外50 ml處做上記號，再將水倒掉。
2. With the valve closed, then transfer the G-50 slurry to the column along the column wall. Be careful to avoid air bubbles trapped in the gel.
將管柱底部的開關關閉，再將Sephadex G-50凝膠溶液沿著管柱邊緣，慢慢倒入管中，不要讓氣泡在管柱內形成。
3. Open the valve to allow buffer flowing by gravity. The G-50 beads will begin to settle, keep adding gel slurry and buffer till the settled beads reached the 50 ml mark on the column. Be careful not to let the gel run dry.
將管柱底部的開關打開，緩衝溶液隨即流出，G-50膠體顆粒會開始沉降在管柱底部，在膠體顆粒還沒有完全沉降前，繼續倒入剩下的G-50膠體溶液，直到沉降的膠體顆粒體積達到50 ml。請注意，絕對不要讓管柱乾掉，膠體的上方一定要有緩衝溶液蓋住。
4. After the column is packed, adjust the flow rate to 2 ml/min.
待管柱填充好，調整流速到2 ml/min。
5. With minimum solution above the gel matrix, add protein sample by gently layered on top of the gel matrix, to keep a flat gel surface.
蛋白樣品要加入管柱前，請先將管柱上的緩衝溶液放掉，待緩衝溶液的高度降到膠體表面時，請馬上將管柱的開關關閉。再將蛋白質樣品溶液，輕輕地加到膠體上方，絕對不要破壞膠體表面的平整，蛋白樣品加完後，請打開管柱底部開關。
6. After the sample sink below the gel matrix, add more buffer to elute the protein mixture.
待蛋白樣品全部進入膠體內，關上開關，慢慢地加入緩衝溶液蓋過膠體表面，同樣的，絕對不要破壞膠體表面的平整。
7. Begin collecting eluent immediately after loading the sample (2 ml/tube).
打開管柱底部開關，開始以每根試管2 ml收集流出液，同時並適當的補充管柱上的緩衝溶液，絕對不要讓管柱乾掉。
8. Measure the absorbance at 280 nm with a spectrophotometer and quartz cuvette (handle with care!!).
利用光度計測量每一根試管收集溶液的280 nm讀值，石英比色管-價錢昂貴請特別小心。
9. Store the fractions of the peak absorbance for gel electrophoresis, (Exp. 5).
將含有蛋白質吸收峰試管的樣品吸取1ml至微量離心管中，標示組別後交至講桌上，以便統一冷凍保存，留待實驗5電泳分析時使用。
III. Results:
1. Plot absorbance at 280 nm against tube number.
請依照試管的管數，與其280 nm吸光度讀值作圖。
2. Look up the molecular weight of ovalbumin, lysozyme, and Vit. B12, and predict the eluted order of these molecules.
請找出雞卵蛋白、溶菌酶、維他命B12的分子量，並預測他們流出管柱的順序。
2.3: Gel Filtration Chromatography is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by LibreTexts.
The book could not be loaded just now.