EasyBlot Kit User`s Manual
Contents
1. Storage instructions 1 EasyBlocker store at 4C 2 EasyBlot anti rabbit IgG CHRP Aliquot and store as a concentrated solution at 20 C or below Avoid multiple freeze thaw cycles2. 29 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 Background EasyBlot anti rabbit mouse IgG is an HRP conjugated secondary antibody that specifically reacts with the native non reduced form of rabbit IgG and does not bind to the reduced denatured form Using Easyblot as a second step reagent 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 Cat No 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 Kit contents 1 EasyBlocker lyophilized Cat No GTX425858 2 GTX225856 01 EasyBlot anti Rabbit IgG Kit Optimized for Prot A G contains EasyBlot anti Rabbit IgG HRP Cat No GTX221666 01 3 GTX225857 01 EasyBlot anti Mouse IgG Kit Optimized for Prot A G contains EasyBlot anti Mouse IgG HRP Cat No GTX221667 01 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 p
3. owder 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 29 GeneTex 2 Dilute the primary antibody with 2 ml of EasyBlocker reagent The incubation conditions will depend on the conditions used for the primary antibody 3 Wash the membrane with TBST buffer 3 times for 5 min each time 4 Dilute the EasyBlot anti rabbit mouse IgG HRP with 2 ml of EasyBlocker reagent and incubate for 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 Rabbit Mouse IgG HRP is 1 1000 Use 2 5 ug of the IP antibody do not exceed 5 ug 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
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