When using phosphorylated antibodies to do WB, what should be noted?

1.Guard against internal enemies

Be sure to add a protease inhibitor to thelysis buffer and add a certain amount of phosphatase inhibitor because thetissue or cell cleaves to release a large number of phosphorylated proteins,the end-to-end protein phosphatase, catalyzes the phosphorylated protein (Rp),resulting in differences that differ from normal physiological conditions.Therefore, the exogenous addition of protein phosphatase inhibitors, inhibitionof phosphorylated protein dephosphorylation, maintenance of proteinphosphorylation state, or even if the band pressure will be very shallow, theresults are not credible.

2. Low temperature allows them to hug eachother tightly

Plus the best after the anti-4 degreesovernight, low temperature allows the antibody and the target protein tightlyhug each other. why? Because the protein antigen on the membrane by thephysical adsorption, easy to fall off at high temperatures. Membrane on theantigen off, the binding efficiency will be low. 4 degrees can also make theprimary defense repeated use several times. After all, phosphorylatedantibodies are expensive. (Soil free) phosphorylation of the protein only asmall part of the total amount of protein, like a party dance so that guestsget together for a couple will have a lot of probability that overnight canensure that antibodies and proteins have sufficient binding time The Secondaryantibody can be room temperature for 1 hour.

3. Which phosphorylated antibody is strong?

Of course it is Cell signaling. His home todo phosphorylated antibodies is good, especially MAPKs phosphorylatedantibodies. It is best to operate the experiment according to the vendor'sprotocol, which is the guarantee of the success of the experiment.

4. They should not "drink milk"

It is recommended to dilute phospho-p38with TBST containing 5% BSA, and the effect is good, rather than using commonTBST containing 5% non-fat milk. Because skim milk is rich in phosphorylatedprotein, it is one of the sources of phosphorus in milk.

5. How to avoid phosphating protein Jun andtotal protein Jun on the membrane "fight"?

After the study of a proteinphosphorylation of the general situation should also look at the total proteinexpression. But the two of the same location, how to do? Can do this: pressureafter the phosphorylation of antibodies, the same membrane to do strip afterthe total protein antibody, and then strip once, and then pressure the internalactin.

6. Listens, yes

When the phosphorylated protein WB is used,there is often a non-specific band in addition to the band of the targetprotein. Phosphorylated antibodies are not good, and even out of non-specificband, and you do not want the band, so after the tablet, be sure to compare themark, you squeeze out the band molecular weight is correct.

7. Washing is also very knowledgeable

Washing the final background of the impactis also larger, millipore recent instructions recommended double distilledwater washing, the effect is obvious, specifically this can be: 1st and 2ndantibodies, DDW 5min X3 times, 2nd antibody, DDW 5min X3 times, TBST 5min X1times, DDW rinse 3-4 times. Comparison of TBST washing membrane, the effect ofa certain gap, the background effectively reduced, even if the tablet 30min,the background is still negligible.

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