Actin Labeling (MSL)

P. Fajer page 1 of 3

Soltions:

Actin prepared from acetone powder (10 mg/ml) GB x 10 FB x 10 0.1M ATP 100mM MSL in DMF (MW = 251) Method:

1. Prepare actin from acetone powder as described in the Methods Book and determine concentration. The concentration should be about 0.250 mM. Actin Volume = V = Actin Concentration +

2. Obtain 100 mM MSL in DMF (stored in Liq. N2) and dilute with FB-ATP to the concentration calculated below to make 1000l. [mM Actin] x 12.5 = [mM MSL] = [mM MSL] (1000l) l of 100mM MSL to add = ------= 100mM 3. Add MSL diluted to above concentration to actin in a ratio of 1:10 (MSL to actin) and let incubate at 0C for 8 to 12 hours. Save aliquot following incubation. l of diluted MSL to add to actin = 1/9 x V = 4. Sediment by centrifugation for 1.5 hours at 200,000xg (70.1 Ti at 51K RPM) at 0C. 5. Wash pellet and sides of centrifuge tube thoroughly with cold FB-ATP to remove any free MSL. 6. Let pellet sit on ice for 10 in a small amount of GB. Carefully scrape the pellet into a Dounce homogenizer with small spatulas. To remove any remaining actin, suspend in a small of GB and remove with a Pasteur pipette. 7. Suspend the actin pellet in FB(DTT) to original volume. Homogenize gently in Dounce homogenizer gently - avoid foaming which is indicative of denaturation. Determine volume #1 | #2? V = | | 8. Clarify by centrifuging in the TJ-6 for 5/max speed/4C. Determine G-actin concentration as in #13. 9. Add 1/9 x V of FB x 10 (ATP) add ATP to 0.2 mM. #1 | #2?

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FB x 10 to add: 1/9 x V = | 0.1M ATP to add: 0.002 x V = | 11. Homogenize immediately while avoiding bubbles. 12. Repeat steps 4-7 and determine the G-actin concentration after step 6 (see step 13) make sure to correct for the FB x 10 addition made in step 7. 13. To determine G-actin concentration: #1 | #2 V = | | D.F. = | A290 = | A320 = | |

 |  [mg/ml] = ------x = | [mg/ml] = ------x = 0.63 | 0.63

Make sure to correct for the FB x 10 addition made in step #7. 14. Determine the amount of MSL bound to actin by double integration of the low power EPR spectrum as compared to a SML standard (0.1 mM). Also check for an unacceptable amount of weakly immobilized component. 15. The MSL actin should be used within a few days of this procedure. Of at least 4 hours each, to remove the FeCN and the rest of the free spin label. e) The FeCN is completely dialyzed out when the yellow color is completely gone (the solution is clear). 10) Check the signal once again on the EPR. If all of the free MSL is gone and there is no weakly immobilized signal, then the labeled S1 can be clarified: centrifuge 15K rpm for 30 minutes at 4C in the SS34 rotor on the Sorvall. 11) Determine the protein concentration: Abs280  Abs320 ------x dilution factor = mg/ml 0.79 Total volume: Total mg = Total volume x mg/ml S1 = ------> SAVE A SAMPLE FOR ATPASES: MSL  S1 AFTER FECN TREATMENT 12) Spins per head: a) Accumulate an EPR spectrum of the final labeled S1: A total of 16 scans (to be averaged) should be accumulated.

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b) Accumulate an EPR spectrum of 0l1 mM free MSL Stqndard, under the same conditions as the Sa (except for gain, which may vary --- the acquisition program will normalize by the gain). c) do the ATPases to determine the fraction labeled (= % SH1 modified: from K-ATPase). d) using the double integral of the free MSL standard, and knowing it’s concentration, determine the amount of spin label in the S1 spectrum. e) Divide the [spins] in the S1 spectrum by the moles of S1 present. 13) Specificity: %SH1 modified (from K-ATPase Specificity = ------spins per head This will be expressed as a %.

0989f4507b942ad29368f015535b66bb.doc10/20/1999 04:55:00 PM10/20/1999 04:55:00 PM