Calculate protein molecular mass

Honestly, I want to share my probblem in Lab. to you all and hopping that I can get the answer immediately.

Is there anyone who know how to calculate protein size (kDa) exactly?

I have a data of L-arabinose isomerase amino acid sequence which has to be checked by using SDS-PAGE method. But, it will not give any benefit if I don’t know the size of the protein that I’ve investigated.

As we know, to judge whether our gene over-expression is success or not we should get the band at the appropriate position in acrylamide gel. The position of the band tell us about its protein molecular mass (kilo Dalton or kDa). If we know how kDa’s our protein are (ex. 55 kDa), then we could judge whether our over-expression is success or not. It is claimed succes if the band lay between 43 and 66.2 kDa (Picture 1). Conversly, it is claimed fail if the band doesn’t appear or appear in other position.

ACAI molecular mass (55 kDa) checked by SDS-PAGE (source: Cheng et al., 2010)

And the problem is, I DON’T KNOW HOW kDa’s MY PROTEIN ARE?? So, how could I judge the result??

–well, I tried to calculate it manually by using this formula:

Suggestion I.

average amino acid molecular mass : 11.0 kDa

number of my amino acid in vector: 534

So, my protein mass (rudely) would be: 534 x 11.0 = 58.74 kDa

But, is it the exact mass?? To be honest, I my self doubt with it. So, I try to find another method which is more valid and trustable. And, this is it: I’ve found the trustable method to calculate protein molecular mass.

Suggestion  II. (More valid, trust me!! *-^!!)

Use an online program called ‘protparam

Enter a Swiss-Prot/TrEMBL accession number,  or

Paste your own sequence in the box availabled –> compute parameters

THE RESULT APPEAR….

Result of my protein molecular mass calculation

Pay attention to  the words “Molecular weight”. Those shows in Dalton (Da). You may convert it to kDa (1 kDa=1000 Da).

Well, finally I got the valid mass now. If it worthfull for you, you may follow my suggestion.

Have a good work!!!

PS:

My protein is: local strain Geobacillus stearothermophilus L-arabinose isomerase or GSAI

Total number of amino acid sequence I put to calculate the mass manually are the AA expressed by araA inserted in pET-21b.

Tips mencampurkan bahan-bahan PCR pada tube

Ada beberapa hal yang perlu diperhatikan dalam mencampurkan bahan-bahan PCR, antara lain sebagai berikut:

1. Semua bahan harus diletakan secara terpisah pada dinding tube, kecuali akuades. Hal ini dilakukan untuk menghindari pencampuran bahan sebelum kondisi PCR dioptimasi/ diatur.

2. Bahan-bahan yang terdiri dari DNA template, MgCl2, Taq buffer, dNTP’s, dan Taq polimerase dapat dicampurkan dalam dinding tube selama preparasi bahan PCR.

3. Forward primer dan reverse primer harus diletakan agak berjauhan.

4. Akuades dapat diletakan di dasar tube.