I have a few confusions of using Pierce Fab2 preparation kit. Sometimes, the spin column doesn't look properly compacted after centrifuging( then I centrifuged with high speed more times and somehow looked ok). And fixing and adding full IgG on pepsin agarose tube has made me confused since it is my first time using it. The pepsin with resin get dissolved with the antibody I add, then how it separates the fragmented one, I am not sure if my adding antibody on Pepsin Agarose slurry is wrong. I couldn't find desired Fab2 fragment , separated from the full IgG after following the full procedure. MALDI data shows,there is no full IgG or Fab or Fab2 or Fc signal coming from sample collected from the kit at the end of the procedure.
The kit components are: Sufficient For: 10 antibody samples, each containing 0.25 to 4 mg IgG • Pierce Pepsin Agarose, 1.25 mL • F(ab')2 Digestion Buffer, 120 mL • NAb Protein A Plus Spin Column, 1 mL, 1 column • PBS Packs (each makes 500 mL), 2 packs • IgG Elution Buffer, 120 mL • Zeba Desalt Spin Columns, 7K MWCO, 2 mL, 10 columns • Spin Columns, 0.8mL, 10 columns • Microcentrifuge Tubes, 2 mL, 30 tubes
The used kit link is: https://www.thermofisher.com/order/catalog/product/44988
If any of you are familiar with the process, please let me know. Thank you.
Suhad Mohammed Suhad Mohammad, I already added the link of thermofisher in my question. My question was related to the kit, how to use it properly because I didn't get expected result. You may tell me if you used this kit or any similar one. Thank you
The hinge region of an immunoglobulin monomer (IgG) is readily accessible to proteolytic attack by enzymes. Cleavage at this point produces F(ab')2 or Fab fragments and the Fc fragment. The Fc fragment may remain intact or become further degraded, depending upon the enzyme and conditions used. Proteolytic IgG fragmentation using three different enzymes is discussed below. Traditionally, proteolysis was accomplished in solution using free enzyme. We have developed immobilized enzyme products that enable better control of digestion and efficient separation of reaction-products from the protease. Thus, the diagrams featured below refer to enzyme "resins." Most procedures also include Protein A resin antibody purification steps to separate Fab and Fc fragments.
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