Bio-rad Immun-Star™ AP Chemiluminescence Kits User Manual

Immun-Star™APChemiluminescent ProteinDetection SystemsFor Use With Nitrocellulose and PVDF MembranesInstruction ManualCatalog #170-5010, 170-5011, 170

Water Purity. Use only deionized distilled water to prepareall solutions. In addition, care should be taken to prevent alkaline phosphatase contaminat

Blotting Grade Conjugates. The conjugates supplied byBio-Rad should be used in the concentrations indicated inSection 2.3. Using a conjugate at higher

9unstained standards are detected by incubating withStrepTactin-AP. Because the avidin-AP (or streptavidin-AP) andStrepTactin-AP can be directly added

2.2 Reagent FlowchartSeveral of the solutions you will make are end-use solutionsand are also used to make up other solutions. For example,1x TBS is

11Reagent Flowchart. (Does not include all reagents in this kit.)10x TBSstockNonfatdry milkNonfatdry milkEnd-usewash solution1x TBSTween 20 TTBSTTBSEn

122.3 Working Solutions (based on 200 cm2of membrane; one large blot, or four mini gel blots)This kit has been designed to blot 2,500 cm2of membrane,

13dedicated-use containers to avoid contaminating other experiments.Because of the high sensitivity of the chemiluminescent assay,care should be taken

Antibody Buffer(0.2% nonfat dry milk in TTBS)Add 0.4 g of nonfat dry milk to 200 ml TTBS. Stir until dissolved. Label this solution “Antibody Buffer”.

2.4 Detailed Assay ProcedureNote: Before beginning, read through the entire procedure.1. Antigen application – apply antigen to the membranesurface u

c. Dot-blotting – cut the membrane sheet to the appropriate size. Draw a grid on the membrane with apencil. Wet the dry membrane by slowly sliding the

Table of ContentsSection 1 Preparation ...11.1 Introduction ...

45° angle, into the blocking solution. Gently agitate the solution using an orbital shaker platform and incubate for30 min to 1 hr at RT, or block ove

9. Blot development – remove the membrane and drainexcess liquid without letting the blot dry. Place on a piece ofSaran wrap on a level surface. Pipet

Section 3Troubleshooting3.1 Troubleshooting GuideProbable Recommended Problem Cause Solution1. No reaction or weak a. Exposure time was i. Increase t

Troubleshooting Guide (continued)Probable Recommended Problem Cause Solutionc. Blot was allowed to i. Use heat-dry after incubation sealablewith the c

Troubleshooting Guide (continued)Probable Recommended Problem Cause Solutionii. Antibody titerwas too low.Increase the concentration ofthe antibodyuse

Troubleshooting Guide (continued)Probable Recommended Problem Cause Solutionii. The concentra-tion of the conjugate wasnonsaturating.Increase the conc

Troubleshooting Guide (continued)Probable Recommended Problem Cause Solutionii. Transfer of protein onto themembrane wasincomplete. Staingel to assure

Troubleshooting Guide (continued)Probable Recommended Problem Cause Solution2. High background. a. Exposure time was i. Decrease too long. exposure ti

Troubleshooting Guide (continued)Probable Recommended Problem Cause Solutiond. Wash stringency i. Tween 20 is was insufficient. necessary inwash steps

Troubleshooting Guide (continued)Probable Recommended Problem Cause Solutionii. Use blottinggrade nonfat drymilk blocker,which has beenquality-control

C

Note on Electrophoresis and BlottingEquipmentBio-Rad provides a complete line of electrophoresis, electrophoretic transfer, and microfiltration appara

Appendix 1Total Protein Detection ProcedureRequired ReagentsCatalog # Description170-6533 Avidin-AP, 1 mlor170-3554 Streptavidin-AP, 0.5 ml170-6529 N-

Tris-buffered saline (TBS)(20 mM Tris, 500 mM NaCl, pH 7.5)Add 120 ml of 10x TBS to 1,080 ml of ddH2O. Label this bottle “1x TBS”.Wash solution (TTBS)

1. Wash the membrane in 100 ml 1x BT solution for 10 min.If the membrane has been in a buffer containing amines,repeat the wash two more times.2. Deca

8. Blot development – remove the membrane and drainexcess liquid without letting the blot dry. Place on a pieceof Saran wrap on a flat, level surface.

4006074 Rev DLife ScienceGroupBio-Rad Laboratories, Inc.Web site www.bio-rad.com USA (800) 4BIORAD Australia 02 9914 2800 Austria (01)-877 89 01 B

Section 1Preparation1.1 IntroductionThe Immun-Star chemiluminescent detection system is a sensitive, nonisotopic method for immunodetection of specif

of the antigen in a polyacrylamide or agarose gel, passively bydirectly spotting the antigen onto a membrane, or by vacuumfiltration using a microfilt

Catalog # Description170-5015 Blotting Reagents Pack170-5056 GAR-AP Blotting Starter Kit 170-5057 GAM-AP Blotting Starter Kit1.4 Complementary Produc

Catalog # Description162-0176 Sheets, 20 x 20 cm, 10162-0177 Roll, 26 cm x 3.3 m, 1162-0174 Sheets, 7 x 8.4 cm, 10Blotting Standards161-0306 Biotinyla

Quantity Shelf Description Provided Storage LifeCatalog #170-5012Immun-Star Substrate Pack– 2,500 cm2• Immun-Star chemiluminescent substrate 125 ml 4°

1.6 Safety Instructions1. Read the entire instruction manual before beginning theassay.2. Wear gloves and protective clothing, such as a laboratoryco