10 replies to this topic

[zogene]

Hello

I am wondering about western blotting for tagged protein with different Abs, if I have protein X with any kind of tag lets say His the expected size will be His+X and when I satin it with His Ab, what if I have X Ab does this protein give me the same size or just X size?

many thanks

[Curtis]

x+His size. His is very small.

[zogene]

thanks for replying, no His is not small it is around 15-20 kd I think
regards

[phage434]

A typical His tag is 6 His amino acids at either the C or N terminus. This will have a MW of around 155*6 = 930 larger than the original protein.

[zogene]

hi
I did not understand very well lets say I have pEt vector with his tagged when I induce it alone with IPTG I have about 15-20 kd band so I suppose when I induce my recobinant protein will be x +20, or I did not understand very well?
many thanks I am just new with proteins

[zogene]

by the way can any one put tagged common protein size like c-Myc/ His/GST/S/Flag/HA
and is tagged usually very small or it can be big size? finally sometimes it mentioned this plamid tagged with X which express protein with X kd? how the tagged express as big but the real size is very small?
many thanks

[neuron]

@Zogene, I don't know if I have understood your last comment correctly. But I think you are confused with empty plasmid size. Like Phage said His  mol wt is 155 and if your protein size  is 40 kda , the recombinant protein with His tag will be 40,000+930=40930 da. which is just increase of ~1kda . The pET vector that you are inducing alone does not contain your gene, that is why it gives you 15-20 kda (~136-180 amino acids). you need to calculate from the vector sequence what are these amino acids. I hope I am making sense , please somebody correct me if I am wrong.

[Curtis]

lol, I work on L protein of NDV virus which is 249 kD. For me 15 kD is small.

[zogene]

Hi
thanks again for response, ok I got the whole point but in many cases  I notice that my protein size is bigger than it should be like in X protein size around 20 kd with His which I supposed it should be 20 either so my protin appeared around 40 kd I checked the frame and there nothing with it, that is what I could not explaine it?

many thanks

[neuron]

This is a very common problem, you should check the other thread also-

http://www.protocol-...erent-mobility/

[Curtis]

zogene, on 30 January 2013 - 11:39 PM, said:

Hi
thanks again for response, ok I got the whole point but in many cases  I notice that my protein size is bigger than it should be like in X protein size around 20 kd with His which I supposed it should be 20 either so my protin appeared around 40 kd I checked the frame and there nothing with it, that is what I could not explaine it?

many thanks

Did you read Phage434's reply at all?