SDS PAGE gives an apparent molecular weight and many proteins behave atypical and run at the wrong size. Some proteins might bind more than the statistical amount of SDS, others less. The only way to make sure you have the correct DNA cloned consists in sequencing. Mass spectroscopy is also an option, but if there is any post-transcriptional modification this also might give "wrong" results. In general I suggest whenever you clone something, sequence it completely. I know examples of people who did research on a protein for two years just to figure out that they had a truncation. Such truncation can easily happen if the PCR is not done correctly or the primers chosen have too different of an annealing temperature, etc etc etc.

Even though BSA is used in many ladders, I do not like BSA as it is a somewhat atypical protein with a lot of hydrophobic surface patches that bind fatty acids. It might run atypical actually.

Hi,

Did you sequenced your vector with universal primers out of cloning site?

Hi Srinivas,

I need more details about your experiment. Can you please elaborate on the protocol that you followed.

How many times have you done this experiment?

Did you load all the samples i mean the lysate, ft...and subsequent samples, and you found this band as to be consistently present in all of them?

How did you purify the protein?

I guess your protein would be Nterm or Cterm His tagged, then you can use ant-His(6) antibody to confirm the prescence of the protein.

Hope this helps.

regards,

Kaushik

Hope the sequence is fine and the molecular wt has been estimated correctly (has Mass spect analysis been done) because the appearance on the coomasie gel doesn ot give correct piture of the molecular wt. you can recheck in a 12% gel against BSA as standard which is 66Kda protein

does the prtein has post translational modifications like phosphorylation, the baculovirous system is suitable for this. if so, please check the molecular wts of the unphosphrylated and phosphorylated forms of the protein.

regards

aparna

Prestained markers do not normally run true to size. Run a unstained marker and check again

SDS PAGE gives an apparent molecular weight and many proteins behave atypical and run at the wrong size. Some proteins might bind more than the statistical amount of SDS, others less. The only way to make sure you have the correct DNA cloned consists in sequencing. Mass spectroscopy is also an option, but if there is any post-transcriptional modification this also might give "wrong" results. In general I suggest whenever you clone something, sequence it completely. I know examples of people who did research on a protein for two years just to figure out that they had a truncation. Such truncation can easily happen if the PCR is not done correctly or the primers chosen have too different of an annealing temperature, etc etc etc.

Even though BSA is used in many ladders, I do not like BSA as it is a somewhat atypical protein with a lot of hydrophobic surface patches that bind fatty acids. It might run atypical actually.

Hi All,

You are all amazing! I appreciate your generosity to support other researchers. I am beginning to understand the reason behind the descrepancy in molecular size of my protein. Thanks all.

Srinivas

Just to add something to the Kai's comment, in my experience, in SDS-PAGE basic proteins (histones) and acid proteins run faster than normal, while neutral and hydrophobic proteins run slower.

Srinivas, have you estimated the molecular mass of your protein from the gene or do you know the real mass of the protein? Your protein could be neutral or hydrophobic and to seem bigger. Have you considered the mass of the tag? (His is a big amino acid ).

good luck...and....

please...let us know the end of the hystory!

paola