The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology
The Hebrew University of Jerusalem
Dr. Mario Lebendiker  Tel: 972-2-6586920  FAX: 972-2-6758963

Bacterial Expression Screen - DDM (Dodecyl Maltoside) lysis - 
                            Small Affinity binding

                Adapted from the talk of Dr.Nicola Burgess-Brown (Structural Genomics Consortium,University of Oxford 
                                            Protein Production Workshop- Oxford 6-7th July 2011 

Screen for soluble or insoluble expression

   1)    Spin 1.5 ml aliquots of bacteria sample 5min 10,000rpm 4°C, discharge sup and keep pellet frozen -80°C      
(Recommendation: duplicate or triplicate for each sample to repeat experiment if necessary)

   2)    Disperse cells in 0.75 ml lysis buffer and left in ice for ~20-30 minutes. 
(Recommendation: use 1.3ml lysis buffer for big pellet of ON overinduction
   3)   Froze cells with liquid nitrogen for less than 20 minutes, and then thawed in room temperature water.
   4)   Spin lysates 15 minutes 14000 rpm at 4°C, and separate sup and pellet. Disperse pellet in 0.75 ml of buffer. Keep a 40ul sample of supernatant
PAGE-SDS: soluble proteins; and 40ul sample of pellet: insoluble proteins or unlysed cells (Recommendation: the use of resuspended pellet
instead of total cell sample, has the advantage that you can see if the cells were lysed or not more efficiently)
   5)  Run samples in PAGE-SDS for Coommasie stain (total proteins) and Western using anti His-HRP for detection (specific protein).
        Run samples in pairs of sup-pellet.
        Buffer: Tris 50 mM pH 7.5, 10% glycerol, 0.5M NaCl
Lysis buffer: Buffer + 0.1% dodecyl maltoside + 1 mM PMSF + 0.2 mg/ml lysozyme + 50μg/ml DNAase (prepare fresh!!!)

Small Screen for IMAC binding

   6) Equilibration of IMAC resin: place 50ul beads (100ul suspension) of Ni-NTA agarose beads (or any other commercial beads) in 1.5ml plastic tube.
Wash with 1 x 1.5ml H2O and 2 x 1.5ml buffer (washing: mix, spin 3min 3500rpm, and remove supernatant).

 7) Mix the supernatant of last step gently with the equilibrated resin for 60 min at 4°C
   8) Spin 3min 3500rpm 4°C. Remove supernatant and keep sample of 40µl for PAGE-SDS: unbound proteins
      9) Wash beads with 1ml buffer several times (washing: mix, spin 3min 3500rpm, keep supernatant aside, be careful not to take the resin) up to 
        OD280nm <0.05 or at least three times. Keep sample of 40µl for PAGE-SDS of each washing.
   10) Elute recombinant protein with 2x250ul buffer + 250mM Imidazol  (incubate 3 to 5min each time before spinning 3min, 3500rpm at 4°C). 
         Keep sample of 40ul for PAGE-SDS of each elution. Keep the rest of the sample to check the oligomeric state (aggregation) of the protein by 
         analytical gel filtration (GF)
    11) Resuspend beads in 50ul H2O + 20ul 5x sample buffer. Mix and spin. Keep sample of 40ul for PAGE-SDS: SDS extracted beads.
     12) According to the number of screening samples, run on PAGE-SDS:  
          A)    Crude supernatant; resuspended pellet; unbound, and first elution
          B)    Crude supernatant; resuspended pellet; unbound, washings, elutions, and SDS extracted beads.
          C)    Only first elution
   13) Check oligomeric state (aggregation) of the protein by analytical GF the relevant samples


Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem  Tel: 972-2-6586920  FAX: 972-2-6758963

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