PROTOCOL Apptsis-Inducing Factr (AIF) Prtein Quantity Dipstick Assay Kit 1850 Millrace Drive, Suite 3A Eugene, Oregn 97403 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 30 60 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.
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
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 250-500 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
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 0.1-10 HeLa cells 0.1-10 HepG2 cells 0.05-5 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 12-20 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
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
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. 500 400 300 200 100 R 2 =0.996 0 0 1 2 3 4 5 6 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. 100 75 50 25 0 1:1 Dilutin 1:2 Dilutin 1:4 Dilutin Page 6 f 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