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1. antibody Chemiluminescent Substrates Wash buffer 1 x TTBS Membrane stripping and re probing buffer aRWON gt Storage and Stability Solutions 1 2 and 3 can be stored at room temperature Solutions 4 5 and 6 should be stored at 2 8 C The kit is stable for one year when handled properly Cytomol Unimed 592 10 Weddle Dr Sunnyvale CA 94089 USA Tel 408 992 0400 Fax 877 713 8298 E mail info cytomol com Website www cytomol com 2 Attoglow Western Blot System User Manual NOD 8 9 10 11 12 Protocol Remove blot from the transfer apparatus and soak in transfer buffer Under the hood pour the Millennium Enhancer solution into a new container For a 50 cm blotting membrane use 10 ml Millennium Enhancer and the solution can be re used 4 times without losing its enhancing effect Pick the membrane from the transfer buffer and drain the remaining buffer on the membrane For best results do not let the membrane dry completely Soak the membrane in the Millennium Enhancer solution agitate for 2 min then remove the membrane and submerge it in 1 x TTBS solution Block the membrane with 5 non fat dry milk in 1 x TTBS solution for 30 min at room temperature with agitation Prepare antibody binding working buffer by diluting the supplied 20x antibody binding stocking buffer 20 times with ddH20 Make an appropriate dilution of primary antibody in antibody binding working buffer 10 ml of primary antibody solution volume2. is suggested for a 50 cm membrane Remove the blocking reagent and add the primary antibody solution Incubate the blot with agitation for one hour at room temperature to overnight at 2 8 C Wash the membrane in 1 x TTBS for 5 min repeat 3 4 times Incubate blot with the appropriate HRP conjugate secondary antibody solution diluted with antibody binding working buffer e g 1 5 000 for anti Mouse IgG for 1 hour at RT with agitation Ten ml secondary antibody solution is suggested for membrane size of 50 cm Repeat step 4 to remove unbound HRP conjugate Prepare substrate working solution by mixing equal volume of Substrate Buffer A and Buffer B right before developing 2 ml substrate working solution mixture of 1 ml Buffer A and 1 ml Buffer B is suggested for a 50 cm membrane Incubate blot with substrate working solution for 1 5 min Place membrane between plastic protection sheets or transparent plastic wrap and mount inside a film cassette with the protein side facing up Place a piece of film on top of the membrane let it expose for an appropriate time and develop the film A recommended initial exposure time is 1 min The blot can be re developed if necessary After stripping the blot can be re probed Re developing Method Soak membrane in 1 x TTBS solution at 2 8 C O N On the second day incubate with secondary antibody and apply wash buffer this step is optional add substrate and expose on film Stripping Method Use
3. Attoglow Western Blot System User Manual Attoglow Western Blot System with Millennium Enhancer The most sensitive non isotope Western blot System Introduction Attoglow Western Blot System is an ultra sensitive chemiluminescent Western blot System developed by CytoMol It detects protein at attogram 10 g or yoctomole 107 M levels This kit consists of three parts Millennium Enhancer protein interaction system and detection system Millennium Enhancer is derived from a powerful innovative proprietary technology Unlike conjugated complex or chemiluminescent substrate enhancers Millennium Enhancer works through a novel mechanism by increasing antigen and antibody affinity and accessibility Due to its unique mechanism Millennium Enhancer can work with different antigens and antibodies such as structural proteins Actin enzymes GAPDH or membrane proteins EGFR It could significantly boost Western blot signals and work together with the two other kinds of enhancing mechanisms to achieve multiplied enhancing effects Millennium Enhancer Millennium Enhancer ing 0 1ng 10pg 1pg 0 5pg 0 1pg 10fg 10ag tag ag 10ag 10fg 0 1pg 0 5pg 1pg 10pg 0 1ng 1ng One ng of purified Rabbit muscle GAPDH was serially diluted to 1 attogram and electrophoresis was performed The gel was transferred to a PVDF membrane and then cut in the middle The right section was treated with Millennium Enhancer and the left was untreated Immunoblotting was do
4. ed 592 10 Weddle Dr Sunnyvale CA 94089 USA Tel 408 992 0400 Fax 877 713 8298 E mail info cytomol com Website www cytomol com 4
5. ents Attoglow Western Blot System Catalog Number pkd7112x 1 4 Item Amount Part No 1 Millennium Enhancer 50 ml pkd7112x 1 2 Antibody Binding Buffer 20 x 60 ml pkd7112x 2 3 Attoglow Blocking Agent 20g pkd7112x 3 4 HRP Conjugated Secondary Antibody anti Mouse or anti Rabbit or 55 110 ul pkd7112x 4 anti Chicken or anti Goat 5 Luminescence substrate solution A 25 ml pkd7112x 5 6 Luminescence substrate solution B 25 ml pkd7112x 6 Reagents are sufficient for 1 200 cm membranes Catalog Number pkd7125x 1 4 Item Amount Part No 1 Millennium Enhancer 100 ml pkd7125x 1 2 Antibody Binding Buffer 20 x 120 ml pkd7125x 1 2 3 Attoglow Blocking Agent 40g pkd7125x 1 3 4 HRP Conjugated Secondary Antibody anti Mouse or anti Rabbit or 110 220 ul pkd7125x 1 4 anti Chicken or anti Goat 5 Luminescence substrate solution A 50 ml pkd7125x 1 5 6 Luminescence substrate solution B 50 ml pkd7125x 1 6 Reagents are sufficient for 2 500 cm membranes Attoglow Western Enhancing Kit Item Amount wka72120 Part No Amount wka72250 Part No 1 Millennium Enhancer 50 ml wka72120 1 100 ml wka72250 1 2 Antibody Binding Buffer 20 x 60 ml wka72120 2 120 ml wka72250 2 3 Attoglow Blocking Agent 20g wka72120 3 40g wka 2250 3 Reagents are sufficient for 1 200 or 2 500 cm membrane Items not supplied in Enhancing Kit Primary antibody Secondary
6. h substrate enhancers enhanced signal magnification is achieved Cytomol Unimed 592 10 Weddle Dr Sunnyvale CA 94089 USA Tel 408 992 0400 Fax 877 713 8298 E mail info cytomol com Website www cytomol com 1 Attoglow Western Blot System User Manual Description Components in this kit are prepared with pure chemicals according to our proprietary technology They are used for ultra sensitive chemiluminescent analysis with Western blotting applications This kit provides a complete system for Western blot analysis including a 1 powerful novel proprietary technology Millennium Enhancer which could significantly boost Western blot signals 2 protein interaction system and 3 chemiluminescent detection system The protein interaction system has been optimized to be compatible with Millennium Enhancer to achieve a superior result Four different formats are provided according to the type of secondary antibody that is supplied HRP conjugated anti Mouse anti Rabbit anti Chicken anti Goat IgG Two different sizes are available 1 200 cm and 2 500 cm of membrane Enhancing activity is confirmed in Western blot analysis of GAPDH protein with the corresponding antibody Quality Control Duplicated Western blots containing 10 fg and 10 pg of antigen are detected with anti GAPDH antibody Antigen at 10 fg levels was detected only on the blot treated with Enhancer with similar intensity as antigen at 10 pg level on the untreated blot Compon
7. ne using a primary antibody clone 6C5 and HRP conjugated rabbit anti mouse antibody and chemiluminescent substrates were applied A film exposed for two minutes is shown above 10 g of antigen without treatment and 10 g of GAPDH with treatment gave similar signal strengths indicating a 1000 fold enhancement Fold enhancement varied from a few to a 1000 fold depending on the characteristics of antibodies and antigen used Features e Sensitive detect antigens at atto gram or yoctomole level extremely useful for low copy genes or antibodies with low sensitivity Others kits only reach up to femto gram level Versatile 100 Enhancement on top of all kinds of chemiluminescent substrates Synergistic Synergistic effect with conjugated complex enhancers and substrate enhancers e Cost Effective The most competitive price on the market sufficient for 2 500 cm membrane Listed price is 20 lower than that of other vendors Western blot kits e Compatible Compatible with PVDF nitrocellulose and nylon membranes e Simple Very user friendly non isotope equipment free e Convenient Complete kit contains secondary antibody and chemiluminescent substrate Applications e Detects trace amount of antigen low expression gene products useful when poor affinity antibodies are available e Western and protein arrays where high sensitivity is needed e Combinations with other Western blot enhancers for an example in combination wit
8. oo much antigen Too much HRP in the system 2 Kit specific Problems 2 1 Little or no Enhancing Effect The antigen level was too high the signal was already saturated Too much buffer remained on the membrane before Millennium Enhancer treatment or Millennium Enhancer has been re used too many times Millennium Enhancer has been diluted too much and lost its activity The membrane was not treated long enough in Millennium Enhancer solution Specific nature of antigen 2 2 High Background A general problem see above The signal was magnified too highly use the kit without Millennium Enhancer treatment Related Products Western Blot Protein array Total protein Compartment Proteins Appendix Preparation of solutions not supplied with kit Solution Preparation Stability temperature Notes 1x TTBS Add 6 05 g Tris base 50 mM 8 76 g 3 months at RT Do not use sodium azide sodium chloride 150 mM to 800 ml distilled as an antimicrobial agent water adjust pH to 7 5 with HCI adjusted to as it inhibit HRP 1 liter with distilled water Add Tween 20 to 0 1 v v Blocking Weigh 5 g of non fat dry milk and dissolve it Freshly made suggested Can be Stored at 2 8 C Solution in 100 ml 1 x TTBS solution O N Stripping 100 mM Glycine pH 2 7 1 month at RT Mild stripping buffer Buffer M Stripping 62 5 mM Tris HCl pH 6 7 with 2 SDS and 1 month at RT Harsh stripping buffer Buffer H 100 mM 2 Mercaptoethanol Cytomol Unim
9. stripping buffer M if the mild condition is sufficient and use stripping buffer H if more stringent stripping conditions are necessary 2 3 Soak the membrane in stripping buffer and incubate at 50 C for 30 min with occasional agitation incubate for a longer time or raise temperature to 70 C if the membrane is not completely stripped Wash the membrane twice in a large volume of 1 x TTBS for 10 min at room temperature Repeat the immunoblotting procedure from the blocking step Trouble Shooting 1 General Problems 1 1 No signal or weak signal Proteins did not transfer properly to membrane Cytomol Unimed 592 10 Weddle Dr Sunnyvale CA 94089 USA Tel 408 992 0400 Fax 877 713 8298 E mail info cytomol com Website www cytomol com 3 Attoglow Western Blot System User Manual Not enough protein loaded on the gel Target protein degradation occurred due to improper storage of blot The concentration of primary or secondary antibody used was too low The blocking buffer used was not correct and antigen was covered Substrate had lost activity 1 2 High background The concentration of the primary or secondary antibody used was too high Too much protein loaded on the gel Insufficient blocking Insufficient washing The level of Tween 20 in blocking buffer was too low Membrane problems e g PVDF membrane was not wetted thoroughly or dried in processing Transfer buffer been contaminated 1 3 Reverse image on film T
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