PROTOCOL 1850 Millrace Drive, Suite 3A Eugene, Oregon

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1 PROTOCOL Apptsis-Inducing Factr (AIF) Prtein Quantity Dipstick Assay Kit 1850 Millrace Drive, Suite 3A Eugene, Oregn MSA31 Rev.0 DESCRIPTION Apptsis-Inducing Factr (AIF) Prtein Quantity Dipstick Assay Kit Sufficient materials fr 30 r 60 dipstick reactins. Kit Cntents: Item MSA31-30 MSA31-60 Dipsticks Gld-cnjugated antibdy (dried in micrplate wells) 30 wells 60 wells Buffer A* (Extractin buffer) 15 ml 30 ml Buffer B* (Blcking buffer) 1 ml 2 ml Buffer C* (Wash buffer) 1 ml 2 ml Strage: Stre dipsticks and gld-cnjugated antibdy at rm temperature ut f direct sunlight in their prvided cntainers. Bth are stable fr 6 mnths. High humidity cnditins shuld be avided. Stre Buffers A and C at 4 C, and Buffer B at -20 C. *Buffers A, B, and C are interchangeable with ther Quantity Dipstick Assay Kits (MS131, MS133, MS431 MSF31, MSP31). After use, be sure t remve any liquid frm the plate befre string in fil bag with desiccant. INTRODUCTION The Apptsis-Inducing Factr (AIF) Prtein Quantity Dipstick Assay Kit is used fr rapid quantificatin f AIF levels frm human sample material. AIF is a 67 KDa flavprtein lcalized in the mitchndrial intermembrane space. It is a multi-functinal prtein with a vital xidreductase functin, an anti-xidant activity, in additin t an assembly functin in Cmplex I frmatin. Mst ntably, AIF has a majr rle in the caspase-independent apptsis prcess. During apptsis, it translcates t the nucleus after mitchndrial release, leading t chrmatin cndensatin and DNA fragmentatin. Based n the immunlgical sandwich assay, the MSA31 dipstick kit utilizes tw mnclnal antibdies specific t different antigens present n the native AIF prtein. One antibdy is embedded n a nitrcellulse membrane while the ther is gld-cnjugated which gives it a reddish clr (Figure 1). When AIF is present in the sample, a red line appears n the dipstick at the site f the AIF capture mab. The signal intensity directly crrespnds t the AIF level in the sample. The signal intensity is best analyzed using a dipstick reader (MS1000) r may be analyzed by anther imaging system.

2 A) B) Figure 1. Schematics f a develped AIF dipstick and the mab-aif-mab sandwich diagram: (A) A finished dipstick shwing a strng signal fr AIF (bttm band) and the psitive cntrl, a gat anti-muse (GAM) antibdy, which acts as a cntrl fr prper wicking f all cntents. (B) The mab sandwich diagram shwing the interactin between AIF, the nitrcellulse-embedded mab and the gld cnjugated mab. ADDITIONAL MATERIALS REQUIRED Dipstick reader (MitSciences MS1000) r ther imaging system Methd fr determining prtein cncentratin Pipetting devices Prtease inhibitrs Page 2 f 7

3 DIPSTICK ASSAY PROTOCOL A. Sample Preparatin Nte: Samples must be kept n ice. Prtease inhibitrs are nt prvided. We prvide enugh Buffer A (Extractin Buffer) fr preparatin f 20 samples using 500 µl /sample. 1. Tissue Sample Preparatin a. Start with apprximately 25 mg f tissue sample. Add 10 vlumes f Buffer A per micrgram f sample (e.g. if the sample weighs 50 mg, add 500 µl f Buffer A). b. Hmgenize the sample. c. Keep n ice fr 20 minutes, mixing intermittently. d. Spin the cell extract in a micrcentrifuge at 13,000-16,000 rpm at 4 C fr 20 minutes t pellet cellular debris. e. Transfer supernatant t a new tube and determine prtein cncentratin (e.g. BCA assays are recmmended as the detergent in Buffer A des nt affect the reading in the assay). f. Prceed directly t Part B f the Prtcl r freeze samples at -80 C. 2. Cell Culture Sample Preparatin a. Start with a cell pellet. Add 5-10 vlumes f Buffer A t the cell pellet and mix (e.g. if the ttal sample vlume displaces 50 µl f vlume, then add µl f Buffer A). b. Keep n ice fr 20 minutes, mixing intermittently. c. Spin the cell extract in a micrcentrifuge at 13,000-16,000 rpm at 4 C fr 20 minutes t pellet cellular debris. d. Transfer supernatant t a new tube and determine prtein cncentratin (e.g. BCA assays are recmmended as the detergent in Buffer A des nt affect the reading in the assay). e. Prceed directly t Part B f the prtcl r freeze samples at -80 C. Page 3 f 7

4 B. Dipstick Prcedure Generating a Standard Curve The assay is mst accurate with a user established standard curve fr interplatin f the signal intensity. Fllwing the prtein cncentratin ranges as defined in Table 1, generate a standard curve using a psitive cntrl sample. We recmmend perfrming a 1:2 serial dilutin with 1 part Buffer A t 1 part Buffer B in a ttal vlume f 50 μl (See Example Experiment Sectin). Sample Type Suggested Wrking Range (µg) Fibrblasts HeLa cells HepG2 cells Table 1. Suggested wrking range fr different sample types Fr Individual Dipstick Reactins 1. Add 25 μl f Buffer B t each well f gld cnjugated mab as necessary. 2. Allw gld cnjugated mab t re-hydrate fr 5-10 minutes. 3. Nw, add 25 μl f sample in Buffer A fr at ttal vlume f 50 µl and mix t hmgeneity. 4. Gently add a dipstick t the well with the thin end dwn (see Figure 1). The cntents will immediately begin wicking up the dipstick. 5. Wick the entire cntents befre prceeding t the wash step (the psitive cntrl band typically appears within 1-2 minutes. A 50 µl sample shuld wick cmpletely in minutes. Mre viscus extracts may take lnger. 6. Add 30 µl f Buffer C and wick cmpletely. 7. Dry the dipstick(s) befre analysis (~20 minutes t air dry, r ~10 minutes at 37 C). 8. Measure the signal intensity with a dipstick reader (MitSciences MS1000 Dipstick Reader) r ther imaging system, e.g. flat-bed scanner. Page 4 f 7

5 FLOW CHART Fr quick reference nly. Be cmpletely familiar with the previus details f this prtcl befre perfrming the assay. Prepare samples. Prepare standard curve. Add 25 µl f Buffer B t a well with dried, gld-cnjugated mab. After 5 minutes, add 25 µl f sample in Buffer A t the abve well. Mix cntents t re-suspend gld cnjugated mab. Add dipstick t sample well and let sample wick up nt dipstick. Add 30 µl f Buffer C t well and wick cmpletely. Dry dipstick. Measure signal. Page 5 f 7

6 Dipstick Scre - % High End Abs.(x1000) MSA31 EXAMPLE EXPERIMENT In the belw experiment we prvide an example using the MSA31 kit t measure AIF quantity frm HepG2 whle cell extract. Samples were prepared as described in this prtcl. All data were analyzed using MitSciences' dipstick reader (MS1000) and GraphPad sftware. Intra-assay and inter-assay CVs are typically 10%. Step 1. Generating a standard curve Belw we shw a typical standard curve fr the AIF Dipstick using HepG2 cell extract (see Table 1). We suggest using 7 t 8 dipsticks fr cvering the wrking range. In this example, the dilutin series starts with 5 µg f cell extract. We used a ne-site hyperbla best-fit analysis R 2 = HepG2 cell extract ( g) Step 2. Analysis f samples We prepared 1:1, 1:2 and 1:4 dilutins f HepG2 whle cell extract, and measured the AIF dipstick signal intensity with the MS1000 reader. Next, we interplated frm the standard curve and cmpared the Dipstick Scre f the diluted samples t the undiluted sample. As shwn belw, the AIF dipstick accurately measures the decreasing AIF signal with the crrespnding decrease in ttal prtein laded :1 Dilutin 1:2 Dilutin 1:4 Dilutin Page 6 f 7

7 TROUBLESHOOTING GUIDE Signal is saturated It is very imprtant that the amunt f sample used is within the wrking range f the assay (a best fit line fr interplatin as generated with the GraphPad prgram). Therefre, it is crucial t knw the wrking range fr the sample type and avid the regin f signal saturatin (see Table 1). Determinatin f the wrking range is recmmended fr the sample in case f signal saturatin. Signal is t weak This ccurs when the sample lacks measurable amunts f the prtein. T increase signal add mre sample t anther dipstick. Psitive cntrl band des nt appear Make sure that the wicking paper is in cntact with the nitrcellulse membrane. Care shuld always be taken when handling the dipsticks t prevent disruptin f this junctin (see Figure 1). Page 7 f 7