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        Preparation of Tonsil Lysate

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        1685

        Purpse

        The prtcl describes hw t prepare human tnsil lysate fr use in purificatin f ICAM-1,LFA-1,and PNAd.

        Materials

        Safety Equipments

        Lab Cat

        Latex Glves

        Face Mask

        Bench Paper

        Fresh human tnsil tissue

        Parafilm

        Scissrs

        Stainless steel mesh screen (200 mesh=74 mm,Type 316; Tylinter,Mentr,hi)

        RPMI-1640 medium

        Tritn X-100

        Lysis Buffer:

        50 mM Tris-HCl,pH 7.5

        150 mM NaCl

        0.02% NaN3

        10 mg/ml Aprtinin

        10 mg/ml Pepstatin

        10 mg/ml Leupeptin

        1 mM PMSF (Made fresh the day f experiment.Disslve in 100% EtH)

        1 mM Benzamidine

        5 mM Idacetamide

        Whatman Paper

        Centrifuge Bttles

        Funnel

        Squeeze bttle

        Several Beakers

        Hmgenizer (Fisher PwerGen 125)

        50ml Crning tubes

        Ice Bucket & Ice

        Dunce hmgenizer (40ml Pyrex Cat N.7727-40)

        1 liter bttle

        Stir bar

        2 Side-arm flasks

        Prcedure

        All wrk must be dne at 4℃.

        1.Cllect 50 grams f tnsil tissue.(this shuld yield a large amunt f ICAM-1,LFA-1,and PNAd)

        Nte 1: Cllecting this much tnsils can take sme time.S it is gd t cllect frm mre than ne surce.

        Nte 2: Yu can prep each sample as yu receive it r wait until yu have a ~ 50g f tnsils.

        If yu decide t prep them right away then perfrm steps 2 t 4 and then freeze at -80℃.

        If yu want t wait until yu have ~50g f tnsil,yu shuld immediately freeze tnsils at -80℃.

        2.Clear a wrk area in the 4℃ walk-in fridge.Lay dwn bench paper and assemble all yu materials.

        3.Mince the tnsils with the scissrs.Try and make the pieces a small as pssible.

        Nte: Yu can use a small glass plate as a cutting surface t make the jb easier.

        4.Fld the wire mesh in the shape f a cne and place in a funnel.Place funnel n the muth f the beaker,t cllect RPMI-1640.

        5.Pur RPMI-1640 int a squeeze bttle.Wash the minces tnsils with cpius amunts f RPMI-1640.Wash a small amunt f tnsils at a time.

        (At this pint yu can freeze the tnsils until yu have enugh)

        6.Set up the hmgenizer with the large prbe (1cm diameter).

        7.Put a small amunt f minced tnsil in the 50ml Crning tube and add lysis buffer t ~10ml mark.

        8.Insert prbe t the tube and the cver the neck f the tube and the main bdy f the prbe with parafilm.This is very imprtant.This prevents aersls frm escaping during the hmgenizing prcess.

        9.Hmgenize in small burst t avid heating the sample.

        Nte 1: Hmgenize till yu liquefy the sample.Yu may still see a little bit f white strands,but these are cnnective tissue and will nt cmpletely break dwn.

        Nte 2 : The prbe will get clgged with cnnective tissue.Clean it f with tissue paper and rinse in with water by pulsing the prbe t remve the rest f the stuck cnnective tissue.nce clean again yu can cntinue hmgenizing yur sample.

        10.Cllect the hmgenized tissue in a beaker that is kept n ice.

        11.Using the dunce hmgenizer,hmgenize the cllected sample using an up & dwn and left & right hand mtin.D this at least 10 times t get a very fine sample.

        Nte: T avid spilling yu sample,dn’t use mre than the vlume stated n the side f the dunce hmgenizer.

        12.When yu are dne transfer hmgenized tissue t a 1 liter bttle and add in the stir bar.

        13.Bring the vlume up t 1 liter by adding mre f the Lysis buffer.

        14.Add Tritn-X100 t a final cncentratin f 2% by vlume.

        Nte: The Tritn-X-100 will nt disslve immediately.It will g int slutin slwly ver night.

        15.Stir gently ver night in the 4℃ walk-in.

        16.Transfer the liquid t centrifuge bttles and spin dwn lysate at 8000g fr 1 hur.

        17.Decant lysate in a clean beaker.Discard pellet.

        18.Filter lysate twice,using Buckner Funnel and Whatman paper as fllws:

        Cut the Whatman paper t cver the bttm f the Buckner funnel.

        Place funnel n side-arm flask and attach.

        Attach the first flask t the secnd and t the vacuum.

        Filter the lysate thrugh the Whatman,changing it ften as it gets clgged.

        19.Filtrate may nw be stred a 4℃ until it is time t run ver an affinity clumn.

        Nte: Dn't stre fr mre than a cuple f days at 4℃.

        References

        K.D.Puri,E.B.Finger,G.Gaudernack,and T.A.Springer (1995).Sialmucin CD34 is the majr L-selectin ligand in human tnsil high endthelial venules.J Cell Bil 131:261-270.

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