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Solutions should be pre-prepared with deionized ultrapure water dH2O or equivalent. These secondary antibodies are provided in solution. When purchasing lyophilized antibodies, resuspend dried antibodies in 1X secondary antibody storage buffer For 10mL mix 4. At this point, the membrane can be evaluated to determine if equivalent amounts of protein were loaded in each lane.
You may wish to photograph or scan the stained membrane or to cut the membrane horizontally so that you can use one primary antibody on the top of the membrane and another on the bottom of the membrane. X Need an answer now? Give us a call Leave a message, we’ll get back to you Your question.
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Protocol To access the protocol, please provide your email address: Combine 91g Tris base with dH2O to a total volume of mL.
Adjust pH to datasueet. Combine 30g Tris base with dH2O to a total volume of mL. Adjust pH to 6. Primary Antibody Dilution Buffer mL: Secondary Antibody Dilution Buffer mL: Depending on the species used to produce the primary antibody: Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Sonicate for 10—15 sec using a probe sonicator to shear DNA.
Quantify total protein content in the lysate using Total Protein Determination Kit Maccording to the manufacturer’s instructions. Fill the chamber up until you reach a point that will be 1cm below the bottom of the gel comb when it is added in the next step. After the separating gel polymerizes usually 15 minutespour off the ethanol. Wash transfer apparatus and sponges well with water immediately prior to use.
A 10mL pipette should be used to gently roll out bubbles after the last blotting paper is laid on the stack. Ponceau S staining protocol.
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Wash nitrocellulose membrane two times with dH2O and then stain the nitrocellulose membrane with Ponceau S Staining Datasehet with gentle agitation. Remove the Ponceau S Staining Solution and wash the membrane with dH2O at least three times and visualize cellular proteins.
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Total Protein Determination kit.
Tris Base Chemzymes Ultra Pure.