SDS PAGE Protein Concentration Estimation

[kiki06]

Please help this person :

I have a problem with my SDS PAGE. I estimate protein concentration by BCA method.I use two dilutions of the protein for protein estimation and take the average to measure the protein concentration of the samples. Yet their a vast variability in the actin loading control fo the samples. How do i get rid of this problem. Can someone please help me with this

vinitha

[danfive]

So the quantitation works fine, but your gels show varying amounts of actin band. It's possible that you have protein degradation, loss to tube stickiness or precipitation, or DNA causing viscosity and thus interfering with accurate pipetting.

Suggestions:

Use a very good lysis buffer, include DTT and/or high SDS percentage.

Use Proteinase inhibitor cocktail and orthovanadate, (maybe even EDTA, like a Tris-EDTA-SDS buffer), if you suspect degradation from proteolytic enzymes.

Clear your protein solution/lysate of insoluble precipitate/cell debris, by centrifuging at 10,000-14000xg for 10min, and transfer clear supernatant to new tube, repeat as necessary.

Aliquot your solution so you avoid multiple freeze-thaw.

Try to avoid storing dilute protein concentrations; better to store at medium to high protein concentration.

If its a lysate pipette up and down to shear DNA and avoid viscosity later on (thanks admin for that tip).

Lastly check your BCA protein quant procedure and make sure its accurate--not using incompatible reagents, and of course your sample's Absorbance value should not fall outside the standard curve (shouldn't be higher or lower than the stds).

[danfive]

Also make sure you use at least 1:1 or 2:1 ratio of loading buffer (Laemmli) to sample, when loading sample into well.