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How many nucleotides should I leave at the 5'-end of my primer for restriction enzymes (RE) to be able to efficiently cut the amplified insert?

Visit this PDF file from NEB that has enzyme-specific information about this:


How to get better ELISA results?

1) Use DPBS instead of PBS for your capture antibody.
2) Filter all of your solutions.
3) Use high-quality BSA from Sigma.

A useful resource:

How to get better phospho-blots?

If you are trying to detect phosphorylation of a protein using a phospho-specific antibody and get an unsatisfactory weak signal, what usually helps is supplementing 1 uM of microcystin-LR to the lysis buffer just prior to cell lysis.

Strong western blot stripping protocol (home-made):

Strong Stripping Buffer: 1% SDS, 63mM Tris-HCl, pH 6.8, 0.5% Tween 20, 0.7% beta-mercaptoethanol (BME).
For 1L: 31.5 ml of 2M Tris-HCl stock, 100 ml of 10% SDS stock, 5 ml of Tween 20, 863.5 ml dH2O. Add BME fresh: 70 ul per 10 ml of the buffer.

1) Rinse the membranes with TBST (5 min, while shaking).
2) Incubate the membranes for 5 min at room temp in 0.2M NaOH.
3) Incubate the membranes for 30-60 min at room temp in Stripping Buffer.
4) Rinse the membranes with TBST (3 times, 5 min each, while shaking).
5) Optional: block as usual.
6) Continue with primary antibody incubations.

Here is a trick I came up with to get cleaner phospho or total blots (if your antibody gives you non-specific bands and/or high background):

1) Run 10-12 lanes of the most commonly used cell lysate (e.g. HEK293 lysate): 20-30 ug per lane.

2) Transfer onto a nitrocellulose membrane.

3) Cut out the region (a thin horizontal strip) that corresponds to the MW of your protein.
You will end up with 2 pieces of membrane (unless your protein is >250 kDa or <20kDa and it's just easier to cut off the top/bottom of the membrane).

4) Block the membranes with 5% milk/TBST for 1hr.

5) Incubate the membranes with your antibody solution at 1 ug/ml in milk or BSA O/N at 4C (it's best to supplement the solution with 0.02% NaN3 as a preservative).

6) Use this antibody solution for your actual experiment.

This negative selection will clean out most of the non-specific species present in your polyclonal antibody pool.

Why do we put EDTA into lysis buffer?

EDTA is a divalent cation chelator. There are many enzymes that would not function without their cofactors and in many cases these cofactors are divalent cations such as Mg++. Mn++, Ca++ and Zn++. If a lysis buffer is supplemented with 1-10 mM EDTA, then metalloproteases, nucleases, phosphatases, kinases and several other types of enzymes will be inactive. This is done to preserve the post-translational modification state of the proteins at the moment of cell lysis or to prevent nucleis acids from being degraded.




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