EXTRACTION EXPERIMENTS on COATED VESICLES ogan Gurel Harvard College 1 duly 1984Introduction and Background Preparation of assembly factor (100K and 50K band proteins) involves basically three steps: 1) a series of centrifugations and gradients to isolate crude CV's 2) extraction (sometimes referred to as stripping) of assembly factor from these CV's 3) a series of columns to purify the assembly factor from the extract. Each extraction involves incubating the CV's in an appropriate buffer under suitable conditions and then centrifuging to recover the supernatant. The goal of these extraction experiements was thus to find conditions that would optimize the amount of 100K and 50K assembly factor proteins extracted in the supernatant. This paper presents the results of those experiments.PROCEDURAL NOTES Electrophoresis 5% stacking gel 10% running gel 100-120 volts 60 lamda loadings except for Extraction D (normalized) and Prep mini-gel 5 and 10 lamda marker loadings Centrifugation Beckman Airfuge at 4C 30 minutes 25 psi Pellet Manipulation bottom 50 lamda of supernatant was always discarded pellet was taken up in two step: 1) stirring with closed micropipette 2) micropipetting 12 times at 150 lamda Incubation on Ice bath assembly : fs firfuge tube A ae Ove Control Extraction 100 lambda CV's in buffer A with 100 lambda 1M Tris HCl buffer pH 7.0, giving an effective Tris concentration of 0.5M. 30 minute incubation at room temperature in the airfuge tube.CV Prep -- Mini-gel Obtained 40mg in lml of CV's (Lowry analysis) and diluted with 3 ml of buffer A to give 10 mg/ml. Approximately Img (100 lambda) of CV's will be used for each individual extraction. Ran a minigel comparing these CV's with old markers at loug,25ug, and 50ug loadings. ‘The new CV's are on lanes 3,4,and 5 respectively. Clathrin, 100K, tubulin, 50K and light chains can be identified. The 100K protein appears to be degraded into a number of bands.ACug B25, g uae AB nn) c old CVS CVs acc — oS Sd 1A Sd 125K Sh CV Prep -- Mini-gelExtraction A -- TIME & TEMPERATURE In this experiment extractions on ice at varying time durations were compared with the control extraction (see Procedural Notes). All extractions were in 0.5M Tris-A buffer pH 7.0. 1s, 1P supernatant and pellet, respectively, of control 28, 2P 30 minute incubation on ice 3s, 3P 60" ; wo 4s, 4P 90" ‘ ce 5S, 5P 120 c 2 6s, 6P 150 " oI a Conclusion There appears to be no significant difference between the six extractions. It is recommended, then, that extractions proceed on ice as further degradation of proteins may be prevented.25 2P 58 5° 45 4P 53 SP 6s EP CVE Extraction A 6/21/84Extraction B -- Tris Concentration In this experiment, extractions of varying effective Tris concentrations from 0.25 to 2.5 M were compared with the control extraction. All incubations were for 30 minutes at room temperature in the airfuge tube. 1s, 1P 0.25 effective Tris concentration 2s, 2P 0.5 M a o (control) 3s, 3P 1.OM ” o o 4s, 4P 1.5 M 0 0 5S, 5P 2.0 M cI u 6s, 6P 25M " u a Gel B was loaded incorrectlly; as such the gel was redone as B-2 and all results for this experiment are interpreted from this gel. Conclusion: Extraction of all proteins (including surprisingly, tubulin) improves as Tris concentration increases Optimal 100K and 50K extractions occurs in 5S which was incubated in an effective Tris concentration of 2M in buffer A. However, at this concentration, most other proteins are well extracted including tubulin, hence it might be better to go with an extraction of 1.5M effective Tris concentration.Extraction B 6/22/84 cectone as B2 (ext a) getExtraction B-2 6/24/84Extraction C pH In this experiment, extractions in 0.5M Tris-A buffer were made at varying pH conditions from acidic to basic values, All incubations, in this case, were for 30 minutes at room temperature. 1M Tris buffer was titrated with conc. HCl to give, the appropriate pH solution. is, 1B pH 2s, 2P 38, 3P 4s, 4P 58, 5P 6S, 6P (control) waers8 Conclusions: An interesting pattern may be observed in this gel. 100K protein is sparingly extracted in basic conditions (ph 9.0) while 50K protein is more readily extracted under such conditions and perhaps slightly less extracted at lower pH values. However, optimal extractions of both 100 and 50K occur at pH values of 7.0 and 7.5 (28 and 3S respectively). Extractions seem to follow the pattern: acidic 6.5 100K extracted 7.0/7.5 optimal extraction basic 9.0 50K extracted5 Cvs CK te) 4 aes ta int: Is Uli iP 23 2P 2s ap qs ap S3sP 6 EP * , + s # 3 Extraction C 6/22/84 cvsExtraction D (regular 6 =~ Multiple Spins on Buffer A normalized) In this experiment, the éffect of multiple spins on the extraction of assembly factor was analyzed. An initial incubation in 0.5M Tris-A buffer was followed by centrifugation into supernatant and pellet after which the pellet was taken up in 200 lambda of buffer A and re-centrifuged for 1 to 3 more times. After each spin the pellet was again taken up in 200 lambda buffer A after taking 150 lambda as supernatant. 5S and 5P represent a slight modification of this theme as _two spins were done with the first pellet taken up in 0.5M Tris-A buffer. The diagram on the next page should clarify the procedure followed. All incubations were for 30 minutes at room temperature. is, 12 1 spin (control) 28, 2P 2 spins 3S, 3P 3 spins 48, 4P 4 spins 5S, 5P 2 spins with 0.5M Tris-A buffer. The normalized gel is loaded differently to equalize the different dilutions of supernatant after taking 150 lambda after each spin. Conclusions: From the unnormalized gel we can see that the pellet appears generally the same regardless of the number of spins, although slightly less 100K appears in the pellet after 2 or more spins. In the normalized version of the gel we see little or no difference in the supernatant indicating that successive spins after taking up the pellet in buffer A are ineffective. The revised Extraction D experiments should provide further results and these are reviewed later.i 009 - Sb . ost - se » O0€ - SZ epquet OST - ST sjonbT[e epqueT OST eATsseoons sjuezeuZedns TTY “yw aezgnq epquet 00z up dn uexe3 s3otted Teuts TT¥ yareot vExeo? waeee * x cn sh (2) Z dz {sz sui Ht oor A we ve xoo) \ an Vevooe vEveor 4 Vd yor } & - ) (2 se (22 sz Jd! I Vf MSM bey f wezBetq eanpesorg sutds etdraqnw -- 7 uoTIOeTaNECh cv 1S IP Zs ap 3 3? 45 4? SS SP Cys i Extraction D 6/25/84(Sh BON BA A SA Or IED. Qs Cis iP 25 2p 35 3P 4S

LOM + x“ N\ . / (2s ae 25 2e! (oon 04 ae a) Gs +1002 BA wor Lom “ \ +200aBACs As IS! 2s! 2p' 1s 2s 35 48 P CVs ~se— —— "¢ “ 4 Pt e ‘ Extraction D (Revised) 6/25/84