User:Elizabeth Ghias/Notebook/Experimental Biological Chemistry/2011/09/27

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Objective

To perform dialysis on the protein extracted from the cells.

Description

  1. Find the sequence of the protein by finding the sequences of the following proteins: MBP, Gyr A, Chitin binding domains.
  2. Use a pI calculator (web address shown below) to determine the pI of the protein. The pI was determined to be 7.327
  3. Use a Misonix Ultrasonic Liquid Processor Sonicator to rip the cells open. The two tubes of cells aquired the previous week were divided into a total of four tubes. Each tube was placed on ice and sonicated 3 times for 30 seconds each, with a 30 second break between each round of sonication.
  4. 4 L of 50 mM Tris with a pH of 9 was prepared. It was divided between 2 large containers
  5. Two pieces of Snakeskin pleated dialysis tubing were cut.
  6. Each of the dialysis tubes were filled with two tubes of the protein extracted from the cells.
  7. Each dialysis tube was placed in its own 2 L container of 50 mM Tris.
  8. The containers were placed in the cold environmental storage room overnight.
  9. 1 L of 50 mM Tris (pH 9) and 1 L of 50 mM Tris and 1 M NaCl (pH 9) were prepared and covered with foil for use the next day.


Data

Sequence of Protein Expressed

MBP

MGNHAGKREL NAEKASTNSE TNRGESEKKR NLGELSRTTS EDNEVFGEAD ANQNNGTSSQ DTAVTDSKRT ADPKNAWQDA HPADPGSRPH LIRLFSRDAP GREDNTFKDR PSESDELQTI QEDSAATSES LDVMASQKRP SQRHGSKYLA TASTMDHARH GFLPRHRDTG ILDSIGRFFG GDRGAPKRGS GKDSHHPART AHYGSLPQKS HGRTQDENPV VHFFKNIVTP RTPPPSQGKG RGLSLSRFSW GAEGQRPGFG YGGRASDYKS AHKGFKGVDA QGTLSKIFKL GGRDSRSGSP

CBD

MKLKLTSKLE RLRTSKKGIG ACVLAAGMTA ITMAPQSAYA HGFVEKPSSR AALCSQNYGV LNLNCGNVMY EPQSLEAPKG FPDSGPIDGK IASAGGLFGG ILDQQTSNRW FKNTIKGGVN TFTWKYTAAH STSKWHYYIT KKGWDPNKPL TRAELEPIGT VKHDGSAASN NLTHTINVPT DRNGYHVILA VWDVADTSNA FYNVVDVNLV NNETPDTVAP SQPTELNASK VSANSVEITW KASTDNIGVK EYQVLRNGEV IDTVPGTTFI DKKLKADTEY TYTIKALDSA GNISKESEKL KVKTTHTIPD IEAPTQPKGL HSMGTTSTTV DLMWSPSEDN VGVDHYIVYR ESAGVMNKIG TAANTSFMDK DLKANTSYNY VVTAVDLAGN ESSRSDVLNV TTKSENSAYE KWDARKAYTK GDRVVHEGKV YEAVQNHQGN GDSNWIFALS LWKPVLNK

Gyr A

MTDTTLPPEG EAHDRIEPVD IQQEMQRSYI DYAMSVIVGR ALPEVRDGLK PVHRRVLYAM YDSGFRPDRS HAKSARSVAE TMGNYHPHGD ASIYDTLVRM AQPWSLRYPL VDGQGNFGSP GNDPPAAMRY TEARLTPLAM EMLREIDEET VDFIPNYDGR VQEPTVLPSR FPNLLANGSG GIAVGMATNI PPHNLGELAE AVYWCLENYE ADEEATCEAV MERVKGPDFP TSGLIVGTQG IEDTYKTGRG SIKMRGVVEI EEDSRGRTSI VITELPYQVN HDNFITSIAE QVRDGKLAGI SNIEDQSSDR VGLRIVVELK RDAVAKVVLN NLYKHTQLQT SFGANMLSIV DGVPRTLRLD QLIRLYVDHQ LDVIVRRTRY RLRKANERAH ILRGLVKALD ALDEVIALIL VALATGRAGD ITLDDLHRPE TFFKTFTQTG CTRVQFFEYK QSTLSFGEGT RTGCLSMNS

[1] was used to find the sequences.

pI was calculated to be 7.3276 and the mass of the protein was calculated to be 134897.79824 Da.

[2] was used to calculate the pI and protein mass.

Notes


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