Description - Allied Biotech, Inc
Contents
1. Required only if Chemiluminescent substrate is used Sensitive X ray films are preferred X ray film developing related reagents excluding Chemiluminescence and apparatus such as film cassettes and developer trays or auto film developing machine are also required Il Procedure Flowchart One hour One step Reaction 30 50 min gt Wash 3 5min gt Development 1 5min 1 One hour One step Reaction e Soak both sides of Western transferred or dot blotted membrane with 10ml of WB Enhancer supplied in the kit and gently shake at room temperature for 5 minutes if Chemiluminescence for consequent development is used or 0 3 minutes if DAB for consequent development is used e Mix 5ug of primary antibody and 200ul of WB Probe supplied in the kit or with the recommended amount in the following table add this Antibody Probe Mixture to the above 10 ml Enhancer and continue shaking for 30 50 minutes at room temperature Amounts of Antibody and Probe to be mixed with 10 ml WB Enhance For DAB development For Chemiluminescence development Boe oe gt ea aaa Primary Ab normal 10 20 ug 83 10 ug Primary Ab high affinity Po Saou 8g 200 ul 200 ul 100 200 ul 100 200 ul Tips 1 The addition of the amount of primary antibody is highly depending on its purity and antigen specific affinity If you don t know your primary Ab is high affinitive or not there are two ways to go 1 Treat your primary Ab as a normal one and2. 2 3 times with distilled water 4 re probe the membrane with a lower concentration of WB Probe amp Primary Antibody after washing with Stripping Buffer see step 4 below Using DAB substrate supplied in kits WB R50 WB M50 and WB G50 gt Allied Biotech Inc 3 10075 Tyler Place Suite 19 Yamsville MD 21754 Tel 301 874 0495 Fax 240 465 5802 e Take 100ul of the 100X DAB solution and 100ul of 100X D buffer and mix them with 10ml of distilled water e Incubate this DAB mixture with the membrane from step 2 at room temperature for a suitable time period usualiy 3 10 minutes till desired color blots appear e Wash the membrane with plenty of distilled water then air dry it 4 Optional Blotting with different antibody If needed the membrane after development can be reused for further staining with different primary antibody This procedure will effectively use your valuable Western transferred membrane e Shake the membrane from step 3 with 10 ml of Stripping Buffer Cat 238 51 for 5 min wash it one time with 10ml of PBS buffer and then rinse it with plenty of distilled water Recover the Stripping Buffer for reusing in future e Repeat the above steps 1 to 3 but use different primary antibody at step 1 Application Examples I Detection of unpurified proteins in cell lysate One step WB is the fastest and the simplest Western blot in detection of specific proteins expressed in cells Comparison of One step W
3. B 1 hr to Regular WB 4 hrs in detection of GAPDH proteins in human cell lysate GAPDH me Upper left One step WB using rabbit anti GAPDH polyclonal antibodies i Upper right Regular WB using rabbit anti GAPDH polyclonal antibodies Primary Ab MAb anti GAPDH Lower left One step WB using mouse anti GAPDH monoclonal antibody Lower right Regular WB using mouse anti GAPDH monoclonal antibody Cine s ten WE Reeular WE ee oe Loaded cell numbers lysate from lane 1 to lane 6 are 10K 3K 1K 0 3K 0 1K and 0 01K respectively GAPDH fase Blots were developed with Chemiluminescence Primary 4b PAb anti GA4POH ll Rapid monitoring and semi quantifying target protein existing in purification fractions One step WB provides a very quick and simple way to monitor a target protein during purification Not only is it a sensitive method can detect lt 10 ng dot by DAB or lt 0 1 ng dot by Chemiluminescence but also a method that requires sample amount as small as 1 2ul per dot test Moreover it can be used as a quick semi quantitative assay if a standard with known protein concentration is employed Dilution Sup Ft W E1 E2 E3 E4 amp Eb EF Std i Min 30 minutes semi quantifying target protein P50 flag existing in purification fractions Serially diluted samples left side and standard right 2000 side were dot blotted onto an NC membrane 1ul dot and then detected with EET mouse anti flag by using On
4. CB Aled Bi User Manual One hour One step Western Blot Kits Product Name Catalog Number amp Content WB R51 Kit 245 10 tests WB M51 Kit 245 10 tests WB G51 Kit 245 10 tests WB R52 Kit 225 10 tests WB M52 Kit 225 10 tests WB G52 Kit 225 10 tests WB R50 Kit 190 10 tests WB M50 Kit 190 10 tests WB G50 Kit 190 10 tests B Allied Biotech Inc One hour One step TM Western Blot Kit R With SuperHi ECL Substrate Suitable for Rabbit Primary Antibodies One hour One step TM Western Blot Kit M With SuperHi ECL Substrate Suitable for Mouse Primary Antibodies One hour One step TM Western Blot Kit G With SuperHi ECL Substrate Suitable for Goat Primary Antibodies One hour One step TM Western Blot Kit R With ECL Substrate Suitable for Rabbit Primary Antibodies One hour One step TM Western Blot Kit M With ECL Substrate Suitable for Mouse Primary Antibodies One hour One step TM Western Blot Kit G With ECL Substrate Suitable for Goat Primary Antibodies One hour One step TM Western Blot Kit R With DAB Substrate Suitable for Rabbit Primary Antibodies One hour One step TM Western Blot Kit M With DAB Substrate Suitable for Mouse Primary Antibodies One hour One step TM Western Blot Kit G With DAB Substrate Suitable for Goat Primary Antibodies For R amp D Use Only Enhancer 100 ML 50XWB Probe R51 2 ML 10X Rapid Wash Solution 100 ML SuperH
5. al primary antibodies are preferred Increase Enhancer soaking blot ime before adding WB Probe and primary antibody antibody or WB Probe Reaction Optimize the amount of antibody or WB Probe by dot blot Reduce the development time oo long Contaminated reagents Use clean equipments freshly prepared buffers Wear gloves and use clean forceps or equipments o handle membranes Signal development Dilute the mixed substrate solution with 1 2 volume of distilled water Use reagent too sensitive chromogenic development reagents such as DAB which is less sensitive but produce lower background than Chemiluminescent reagent C3 Allied Biotech Inc 5 10075 Tyler Place Suite 19 amsville MD 21754 Tel 301 874 0495 Fax 240 465 5802
6. do your test If the read out signal accompanies a high background wash the membrane once with Stripping Buffer see step 4 below then restain it with a lower concentrations of Primary Ab or both primary Ab and Probe or 2 Treat your primary Ab as high affinitive and do your test If the read out signal is too weak wash the membrane with distilled water then place it back to the original reaction mixture and restain it after adding additional amount of primary Ab or both primary Ab plus Probe 2 The membrane must be fully covered by solution during shaking 3 If using WB Enhancer taken at refrigerator temperature extend the incubation time for an additional 15 min 4 For convenience the one step reaction can be left overnight at 4 C e f desired recover this used One step Reaction Mixture consisting of Enhancer primary antibody and WB Probe and refrigerate at 2 8 C The One step Reaction Mixture can be subsequently reused 3 4 times for testing the same antigen 2 Wash e Prepare 1X Rapid Wash Solution Shake the bottle of 10X Rapid Wash Solution as it might contain precipitate for 10 sec then dilute the solution with 9 times volume of distilled water e Briefly rinse the membrane from step 1 with plenty of distilled water to remove free antibody and WB Probe e Wash the membrane for 2 minutes with 30 ml of 1X Rapid Wash Solution by aggressively shaking to remove non specific binding substances Rinse the membrane again with
7. e of a typical regular Western blot contains seven steps blocking washing primary antibody binding washing secondary antibody binding washing and developing and requires 4 5 hours In contrast the procedure of One hour One step WB only contains one step reacting washing and developing and requires as short as 30 minutes Currently we provide twelve types of One hour One step WB kit as shown on the above table Each kit is sufficient for detecting 10 without reuse to 40 if reused mini gel size membranes Application for R amp D use only Rapid Immuno blot Western blot Dot blot for Rapidly identifying a specific protein either purified or unpurified Rapidly detecting a specific protein expressed in cells Rapidly monitoring a target protein in a purification procedure just using a small amount of samples 2ul Rapidly semi quantifying a specific protein using just 2u of sample Rapidly screening or titrating antigen specific antibodies Features NO O1 B WD Storage Easy a simple one step reaction a regular procedure needs 4 step blocking 1st antibody binding washing and 2nd antibody binding Rapid the whole procedure takes less than one hour a regular immuno blot takes 3 5 hrs Universal suitable for most primary antibodies Sensitive comparable sensitivity with regular immuno blot Reusable antibodies and reagents can be reused Restainable membrane can be re probed with different ant
8. e step WB kit M a rapid 30 min incubation Blots were developed with DAB gee 74 Sup Supernatant before loading to column Ft Flow through W Wash fraction E1 E7 Eluted fractions 1 7 Std Standard purified P50 flag with known concentrations indicated on the left side Ill Rapid Titration of Primary Antibody One step WB offers a fast and simple means to titrate primary antibody to determine the optimal concentration of primary antibody for Western blot The method is also suitable to examine antibodies from various sources for selection of the best antibody 30 minutes dot blot titration of primary antibody Six serially diluted antigen were dot blotted 1ul dot onto six NC membranes 1 5cm x 2 5cm as indicated on the right panel Membranes were incubated for 30 minutes with 2ml Enhancer containing 40ul WB Probe R and one of he following amounts of rabbit polyclonal antibodies 0 5ul panel 1 1ul panel 2 2ul panel 3 3ul panel 4 4ul panel 5 and 6ul panel 6 Blots were developed with DAB CD Allied Biotech Inc 4 10075 Tyler Place Suite 19 Famsville MD 21754 Tel 301 874 0495 Fax 240 465 5802 Troubleshooting Guide Signal is weak or Too little protein is used Load more protein s onto the SDS PAGE gel invisible Poor Western transfer Optimize transfer time and or the electrical current Make sure that there are no air bubbles between the membrane and gel Wrong type of primary Check the
9. i Chemiluminescence 40ML User Manual One copy Enhancer 100 ML 50XWB Probe M51 2 ML 10X Rapid Wash Solution 100 ML SuperHi Chemiluminescence 40ML User Manual One copy Enhancer 100 ML 50XWB Probe G51 2 ML 10X Rapid Wash Solution 100 ML SuperHi Chemiluminescence 40ML User Manual One copy Enhancer 100 ML 50XWB Probe R52 2 ML 10X Rapid Wash Solution 100 ML Chemiluminescence 40ML User Manual One copy Enhancer 100 ML 50XWB Probe M52 2 ML 10X Rapid Wash Solution 100 ML Chemiluminescence 40ML User Manual One copy Enhancer 100 ML 50XWB Probe G52 2 ML 10X Rapid Wash Solution 100 ML Chemiluminescence 40ML User Manual One copy Enhancer 100 ML 50XWB Probe R50 2 ML 10X Rapid Wash Solution 100 ML 100X DAB Solution Set 2 ML User Manual One copy Enhancer 100 ML 50XWB Probe M50 2 ML 10X Rapid Wash Solution 100 ML 100X DAB Solution Set 2 ML User Manual One copy Enhancer 100 ML 50XWB Probe G50 2 ML 10X Rapid Wash Solution 100 ML 100X DAB Solution Set 2 ML User Manual One copy 10075 Tyler Place Suite 19 Ijamsville MD 21754 Tel 301 874 0495 Fax 240 465 5802 Product Description One hour One step Western Blot Kit patent pending is an innovative and the most advanced Western blot kit By using the kit your Western blot will be no longer a time consuming and labor intense procedure The procedur
10. ibodies Reproducible result is highly reproducible Wash Solution solution set SuperHi or Non mens ican store at room 1 year 1 year temperature Note Kit will be shipped in ambient temperature bn Comparison of One hour One step WB with Regular WB Cne step WE oa sd gt Ore 3 tep a Color a Primary Ab y aed dh z Bloc inding indig Regular WE i Forgetit gt Deve loperent Protocol I Additionally required materials not supplied in the kit Primary antibody Purified monoclonal or affinity purified polyclonal antibodies antigen specific IgGs are preferred Although unpurified antibody or serum is also suitable for the One step WB user may need to optimize both concentrations of antibody and antigen to avoid producing a poor ratio of signal to noise CD Allied Biotech Inc 2 10075 Tyler Place Suite 19 Yamsville MD 21754 Tel 301 874 0495 Fax 240 465 5802 Note We currently provide different types of One hour One step WB kits Kits labeled with R are suitable for using together with rabbit primary antibodies with M suitable for using together with mouse primary antibodies and with G suitable for using together with goat primary antibodies Western blot Membrane Normal Western blot membranes such as Nitrocellulose or PVDF membrane are suitable for One step WB User must perform PAGE and Western transferring ahead of using this protocol X ray film amp its related reagents
11. label of the Kit and select a correct primary antibody compatible with One antibody or One Step WB Step WB Kit The kits labeled with R M and G are suitable for using with rabbit Kit mouse and goat primary antibodies respectively Poor specificity in Use high affinity and purified monoclonal or affinity purified polyclonal primary binding activity of the antibodies primary antibody Primary antibody diluted Increase the concentration of the primary antibody Titration of primary antibody by oo much Dot blot using serially diluted samples from 1 yg ul to 1 ng ul and serially diluted primary antibody is highly recommended to optimize primary antibody concentration Primary antibody or Check Expire Date of the antibody as well as the One step WB kit Use fresh or One Step WB Kit is unexpired antibody or kit defective Incubation time too short Increase One step Reaction time In most cases 30 minutes to one hour incubation or the reagent has not at room temperature is enough However when the antigen amount to be detected is been warmed up ery small or primary antibody is weak increasing incubation time is helpful Also if solutions stored in a refrigerator are used before being pre warmed to RT longer incubation time is needed High background Non specific binding and Select antigen specific high affinitive primary antibodies Purified monoclonal or non specific bands on cross reactivity of primary jaffinity purified polyclon
12. plenty of distilled water e Repeat the above wash and rinse for another 1 to 2 times repeat at least twice if SuperHi chemiluminescence is used for development 3 Development Using normal Chemiluminescence or SuperHi Chemiluminescence supplied in kits WB R52 WB M52 and WB G52 or WB R51 WB M51 and WB G51 Place the water rinsed membrane from step 2 in an X ray film cassette protein side up e Mix 2 ml Solution A and 2 ml Solution B of the chemiluminescent substrate and drop 0 5 ml of this developing solution onto the membrane to cover the whole protein side e Cover the membrane from one end to the other with a transparent film or clear Sara Wrap When covering the membrane press the cover sheet in order to remove bubbles and excessive developing solution between cover sheet and membrane e Transfer the cassette to a dark room and expose an X ray film on the covered membrane protein side facing the X ray film for a suitable time period usually 10 seconds to 5 minutes It is recommended to expose multiple X ray films for different time periods e Develop the X ray film in darkness or use an automatic developing machine Tips SuperHi Chemiluminescence is a very sensitive substrate If developed X ray films are too dark perform one or combination of the following actions 1 simply reduce the exposure time to as short as 1 second 2 extensively wash the membrane for another 5 min 3 dilute the mixed A amp B developing solution
Download Pdf Manuals
Related Search