Easy-WESTERN
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1. 3 Beacle Easy WESTERN II Super Primary Antibody Detection Reagent for Western Blots User Manual for High Sensitivity and Strong Signal Detection Immediately after receiving the kit read the section titted COMPONENTS AND STORAGE It is IMPORTANT to store reagents under proper storage temperatures to prevent inactivation of reagents This manual is for the following kits ver 1 00 Cat Product Name Components BCL EZS21 Easy WESTERN II Super basic MAD reagent Dilution buffer BCL EZS22 Easy WESTERN II Super Marker detector set MAD reagent Dilution buffer Marker Detector BCL EZS23 Easy WESTERN II Super full set MAD reagent Dilution buffer Mouse IgG Enhancer Marker detector BCL EZS24 Easy WESTERN II Super Mouse enhancer set MAD reagent Dilution buffer Mouse IgG Enhancer The kit and its components are for RESEARCH USE ONLY not for diagnostics or medical purposes B Bridge International distributor www b bridge ccom 408 252 6200 customersupport b bridge com Bs Beacle er INTRODUCTION Easy WESTERN EZW is a primary antibody detection reagent kit for Western blots The kit is based on the Multi Antibody Detection MAD technology The MAD reagent is nano size protein particles with high affinity to antibodies Each particle is composed of about 100 antibody binding proteins and is labeled with 50 HRP molecules Because of the2. S s B Ox 2 Beacle 1 ING ver 1 00 10 Wash membrane with TBS T for 5 minutes Repeat 4 more times for a total of 5 washes 11 Detect signal with commercially available HRP substrate TROUBLE SHOOTING B Bridge International distributor Problem Possible Solutions Increase antigen concentration Increase primary antibody concentration Increase the electric current or transfer time to improve protein transfer to membrane Over blocking can reduce signal intensity Reduce the blocking time or lower the Weak signal concentration of blocking agents Primary antibody is either mouse IgG or Goat IgG Consider using kits BCL EZS03 or BCL EZS04 which contains Mouse Enhancer for improved signal detection of mouse IgG EZS kits do not work well with goat IgG When diluting MAD Reagent in buffer without blocking agents use low protein binding tubes White out of Too much antigen or antibody Too much signal inhibits luminescence Reduce the luminescent signal concentration of antigen or antibody used Too many extra bands Non specific binding of primary antibody Reduced the antibody to appropriate concentration Too much protein Reduce the amount of protein in electrophoresis Too high a concentration of MAD Reagent Reduce MAD in reaction Insufficient blocking Block the membrane with 5 skim milk in TBS T for over 1 hour MAD reagent is inactivated due to inappropriat
3. first running a test blot to determine the best dilution factors of each antibody ASSAY PROTOCOLS STANDARD PROTOCOL This method is for high sensitivity and strong signal 1 Separate protein sample s using SDS PAGE 2 Transfer protein to PVDF membrane 3 Block with blocking solution for 1 hour at room temperature RT 4 Wash membrane with TBS T for 5 minutes Repeat 2 more times for a total of 3 washes 5 Incubate the membrane with primary antibody in 0 1x Dilution Buffer with TBS T for 1 hr at RT Primary antibody should be diluted to manufacturer s specifications 6 Wash the membrane with TBS T for 5 minutes Repeat 2 more times for a total of 3 washes 7 Dilute the MAD reagent 1 2 000 in 1x Dilution Buffer and incubate membrane in the solution for 1 hr at RT To get stronger signals use MAD reagent at a 1 1 000 dilution a If mouse IgG is used for the primary antibody dilute the Mouse Enhancer reagent 1 2 000 in the 1x Dilution Buffer containing MAD The Mouse Enhancer reagent is included in kits BCL EZS23 and BCL EZS24 b If molecular weight markers such as MagicMark XP are used dilute the Marker Detector reagent 1 10 000 in the 1x Dilution Buffer containing MAD The Marker Detector reagent is included in kits BCL EZS22 and BCL EZS23 8 Wash membrane with TBS T for 5 minutes Repeat 2 more times for a total of 3 washes 9 Detect signal with commercially available HRP substrate REPROBING PROTOCOL This method
4. water 3 Membrane blocking reagent such as BSA based casein based blocking reagents and reagent grade skim milk Skim milk may give a weaker signal compared to other blocking reagents 4 HRP substrate such as DAB for chromogenic detection or luminol based for chemiluminescence REAGENT PREPARATION 1 Prepare TBS T or purchase readymade 2 Dilute 10x Dilution Buffer 1 10 with distilled water This will make a working stock of 1x Dilution Buffer for B Bridge International distributor www b bridge com 408 252 6200 customersupport b bridge com the MAD reagent 3 Dilute the 1x Dilution Buffer 1 10 with TBS T This will make a working stock of 0 1x Dilution Buffer with TBS T for the primary antibody 4 Prepare membrane blocking solution according to manufacturer s instruction or 5 reagent grade skim milk in 1x Dilution Buffer 5 To detect molecular weight markers that are typically detected by secondary antibodies such as MagicMark XP use Marker Detector reagent provided with kits BCL EZS22 and BCL EZS23 Dilution instructions provided with each protocol below 6 To enhance weak signals from mouse IgGs such as IgG1 use the Mouse Enhancer reagent provided with kits BCL EZS23 and BCL EZS24 Dilution instructions provided with each protocol below IMPORTANT When multiple antibodies are used with the MAD reagent to probe a membrane signal may be reduced due to binding competition of antibodies to the MAD reagent We recommend
5. blocking solution for 1 hour at room temperature RT Wash membrane with TBS T for 5 minutes Repeat 2 more times for a total of 3 washes Incubate the membrane with primary antibody for 1 hr at RT The primary antibody should be diluted to the manufacturer s specifications in any buffer you usually use Wash the membrane with TBS T for 5 minutes Repeat 2 more times for a total of 3 washes Incubate the membrane in the 2 antibody conjugated with HRP for 1 hr at RT The secondary antibody should be diluted to the manufacturer s specifications in any buffer you usually use Wash membrane with TBS T for 5 minutes Repeat 2 more times for a total of 3 washes Dilute the MAD reagent 1 2 000 in 1x Dilution Buffer and incubate membrane in the solution for 1 hr at RT To get stronger signals use MAD reagent at 1 1 000 dilution a If mouse IgG is used for the primary or am antibody dilute the Mouse Enhancer reagent 1 2 000 in 1x Dilution Buffer containing MAD The Mouse Enhancer reagent is included in kits BCL EZS23 and BCL EZS24 b Generally the Maker Detector reagent is not needed when a ana antibody is originally used to probe the membrane If a stronger marker signal is needed dilute the Marker Detector reagent 1 10 000 in1x Dilution Buffer containing MAD The Marker Detector reagent is included in kits BCL EZS22 and BCL EZS23 B Bridge International distributor www b bridge com 408 252 6200 customersupport b bridge com oe
6. e storage MAD Reagent should be stored at 20 C Inactivated MAD can produce non specific signals Replace MAD Reagent High background Insufficient washing Increase the number and the duration of washes Adequate signal but with high background decrease primary antibody concentration and or decrease incubation time Reduce the concentration of MAD Reagent When using antigen antibody reaction enhancers insufficient washing causes high background Increase the number and the duration of washes Weak signal of 1 antigen when detecting multi antigens One primary antibody weakly binds to antigen or MAD Reagent Increase the concentration of the primary antibody giving the weak signal Insufficient washing of primary antibody Increase the number and the duration of washes www b bridge ccom 408 252 6200 customersupport b bridge com B Beacle rer Related products Product Product name Description BCL EZM01 Marker detector For Easy WESTERN Kits 50 test BCL EZE01 Mouse IgG enhancer For Easy WESTERN kits 50 test BCL EZB21 10x Dilution buffer For Easy WESTERN Kits 60mL BCL 125A Signal Booster Solution A Enhancer for antibody antigen reaction 250 mL B Bridge International distributor www b bridge com 408 252 6200 customersupport b bridge com
7. is to enhance weak signals without stripping the membrane to reprobe B Bridge International distributor www b bridge com 408 252 6200 customersupport b bridge com Bt Beacle 27 0k MR The membrane must still be wet with buffer from the original probing method Dried membranes cannot be used Wash membrane with TBS T for 5 minutes Repeat 2 more times for a total of 3 washes Dilute the MAD reagent 1 2 000 in 1x Dilution Buffer and incubate membrane in the solution for 1 hr at RT To get stronger signals use MAD reagent at a 1 1 000 dilution a If mouse IgG is used for the primary or pm antibody dilute the Mouse Enhancer reagent 1 2 000 in 1x Dilution Buffer containing MAD The Mouse Enhancer reagent is included in kits BCL EZS23 and BCL EZS24 b Generally the Maker Detector reagent is not needed when a on antibody is originally used to probe the membrane If a stronger marker signal is needed dilute the Marker Detector reagent 1 10 000 in 1x Dilution Buffer containing MAD The Marker Detector reagent is included in kits BCL EZS22 and BCL EZS23 Wash membrane with TBS T for 5 minutes Repeat 4 more times for a total of 5 washes Detect signal with commercially available HRP substrate ENHANCED SIGNAL USING 2 ANTIBODY PROTOCOL This protocol is designed for using MAD to enhance signal from a au antibody HRP a fF Oo N gt Separate protein sample s using SDS PAGE Transfer protein to PVDF membrane Block with
8. se properties MAD reagent enables high sensitivity and quick detection of primary antibodies The Easy WESTERN kit is ideal for high sensitivity signal enhancement and simultaneous detection of multi antigens that is not possible with standard Western blot techniques Advantages 1 No need for secondary antibody MAD reagent can detect most primary antibodies 2 Higher signal for weakly expressed antigens 3 Enhance signal by easy reprobing no stripping the membrane 4 Improve signal while using less primary antibodies MAD reagent may not work well with goat IgG For best results use Mouse IgG Enhancer with mouse IgG1 primary antibodies The performance of EZW depends on the type of antibody and we do not warrant higher sensitivity in all cases COMPONENTS AND STORAGE 1 Multi Antibody Detection MAD Reagent 250uL Store at 20 C immediately upon receipt and after every use 2 10x Dilution Buffer 60mL Store undiluted buffer at 20 C or diluted at 4 C 3 Marker Detection Reagent 50 L kits BCL EZS22 BCL EZS23 Store 20 C 4 Mouse Enhancer Reagent 250 L kits BCL EZS23 BCL EZS24 Store 20 C Marker Detection Reagent and Mouse Enhancer Reagent are provided in an antifreezing solution They do not freeze at 20 C All components should be stored at the recommended temperatures to prevent inactivation REAGENTS NEEDED NOT PROVIDED 1 TBS T 150mM NaCl 10mM Tris HCl 0 1 Tween 20 pH 7 6 2 Distilled
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