EasyBlot Kit User`s Manual
Contents
1. EasyBlot anti Mouse IgG Kit GTX221667 01 GTX425858 Optimized for Prot A G EasyBlot anti Mouse IgG HRP EasyBlocker GTX225857 01 EasyBlot anti Rat IgG Kit GTX628474 01 GTX425858 Optimized for Prot A G EasyBlot anti Rat IgG HRP EasyBlocker GTX228909 01 EasyBlot anti Goat IgG Kit GTX628547 01 GTX425858 Optimized for Prot A G EasyBlot anti Goat IgG HRP EasyBlocker GTX228910 01 EasyBlot anti Sheep IgG Kit GTX628906 01 GTX425858 Optimized for Prot A G EasyBlot anti Sheep IgG HRP EasyBlocker GTX228911 01 3 GeneTex Preparation of EasyBlocker reagent 1 Keep the EasyBlocker powder at RT for 10mins before dissolving it 2 Dissolve the blocking powder in TBST buffer 0 5 g powder in 10 ml TBST buffer Shaking it for 30 40 mins at RT to make sure that it is dissolved completely Note In order to conserve the EasyBlocker reagent we recommend diluting the antibody in 2 3 ml of EasyBlocker reagent and incubating the transferred membrane in a sealed bag Make sure to remove as much air from the bag as possible 3 We recommend that you reconstitute the EasyBlocker reagent immediately before use Protocol 1 After transferring the IP sample to a PVDF or NC membrane block the membrane with 5 ml of EasyBlocker reagent for 1 hr at room temperature 2 Dilute the primary antibody with 2 ml of EasyBlocker reagent The incubation conditions will depend on the conditions used for the2. amp GeneTex EasyBlot Kit User s Manual GTX225856 01 EasyBlot anti Rabbit IgG Kit Optimized for Prot A G GTX225857 01 EasyBlot anti Mouse IgG Kit Optimized for Prot A G GTX228909 01 EasyBlot anti Rat IgG Kit Optimized for Prot A G GTX228910 01 EasyBlot anti Goat IgG Kit Optimized for Prot A G GTX228911 01 EasyBlot anti Sheep IgG Kit Optimized for Prot A G Background EasyBlot is an HRP conjugated second step reagent that specifically reacts with the native non reduced form of IgG and does not bind to the reduced denatured form Using Easyblot decreases the interference caused by the heavy 50 kDa and light chains 25 kDa of the IgG used for immunoprecipitation IP The protein A G beads used in the IP procedure sometimes bind to IgG and produce contaminating signals at approximately 50 kDa and 25 kDa This kit also includes an EasyBlocker GTX425858 that can minimize the background caused by protein A G contamination GeneTex s EasyBlot kit is simple to use and detects only the target band in your IP experiments resulting in cleaner western blots Note Suggested dilution is 1 1000 Use 2 5 ug of the IP antibody do not exceed 5 ug Completely reduce all IP samples Kit contents Catalogue f Product name Kit contents number EasyBlot anti Rabbit IgG Kit GTX221666 01 GTX425858 Optimized for Prot A G EasyBlot anti Rabbit IgG HRP EasyBlocker GTX225856 01
3. primary antibody 3 Wash the membrane with TBST buffer 3 times for 5 min each time 4 Dilute the EasyBlot anti IgG HRP with 2 ml of EasyBlocker reagent and incubate for 1 hr at room temperature 5 Wash the membrane with TBST buffer 3 times for 5 min each time 6 Develop the signal with ECL ECL reagent and detect the signal using either X ray films or a digital camera Note The recommended dilution ratio for the EasyBlot anti IgG HRP is 1 1000 Use 2 5 pg of the IP antibody do not exceed 5 pg Completely reduce all IP samples by 100mM DTT Recipe 20X TBS buffer 1 L Tris 48 4 g NaCl 160 g Add ddH20 to 1 L Mix to dissolve and adjust the pH to 7 0 using concentrated HCl TBST buffer 1 L 20X TBS buffer 50 ml Tween 20 2 ml Add ddH20 to 1 L Adjust pH to 7 0 if necessary GY GeneTex Storage instructions 1 EasyBlocker store at 4 C 2 EasyBlot anti IgG HRP Aliquot and store as a concentrated solution at 20 C or below Avoid multiple freeze thaw cycles
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