sds page result

[Gayathri Selvaraju]

I'm a student and currently doing my final year project on "Partial purification and characterization of thermostable alkaline protease". I did sds page but I only managed to get the standard protein marker band. But, not my sample's band. May i know what's the reason for this situation. And protein content of my sample is very low. So may i knw what is the reason for this. Moreover, if i included this in my result, how am I suppose to discuss my result?

Thank you

[mdfenko]

how did you visualize? coomassie?

since you have very little sample you should use a very sensitive detection method (eg silver stain).

how did you prepare your sample?

what is the size of your protein?

what percentage gel?

we need more information to make recommendations.

[Gayathri Selvaraju]

I stained by using coomasie .
k just now when I repeated the experiment, i managed to get my sample bands. but it is too light and even couldn't detect the bands.
the gel i used was 12.5%.
the molecular weight of my protein is around 35kDa.
SDS PAGE is recommended by here. so that's why i did not use silver staining.
i prepared my sample with 2X sample buffer.
earlier i extracted my crude protein through centrifugation.
I used heat treatment to for partial purification of my sample. The protein content is too low in the range of 0.0001 like that.

[mdfenko]

you can stain sds-page with silver. it is a lot more sensitive than coomassie.

with a low concentration protein, you use a larger volume and the salts of the sample's buffer can cause distortion. you also may have insufficient stacking. this will allow the band to broaden beyond an easily detectable concentration.

[relaxin]

If you are not limited by the amount of starting material, you can concentrate your protein by acetone precipitation. Just add 5 volumes of cold (-20 C) acetone to 1 volume of your sample, mix well, and centrifuge down the precipitate, air dry pellet and redissolve in small amount of sample buffer.

[Gayathri Selvaraju]

Hi,
I used 2X sample buffer which contains 20% glycerol. Suppose I should use 5% glycerol. Besides that, I run the sds page at 20mA. But, the current dropped to 9mA half away. Even, the running buffere were full. But, still I managed to get bands for my protein marker, not for my samples.

[relaxin]

If you mix equal volume of sample and 2X sample buffer, you will get a final concentration of 10% glycerol. This should not pose a problem, since the main function of glycerol is to make the sample dense. You do not get band(s) with sample, because the protein concentration is too low.

[Gayathri Selvaraju]

Thank You for the replies...

[Gayathri Selvaraju]

I tried protein precipitation by using acetone, but my protein become too hard and difficult to resoluble in buffer...may i know the reason?

[mdfenko]

a tight pellet will take more work to resolubilize.

you can use a pestle to homogenize.

if using sds-page sample buffer to dissolve the pellet, as recommended by relaxin, then you can heat the sample.