How to interpret a Western Blot? - (Mar/04/2007 )

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Hi Bioforumers,

I'm new to biology and was just wondering how would I interpret a Western blot? I have grown and expressed T. cruzi Trans-sialidase in 100ml culture. When an OD of 0.6 was reached I induced with IPTG and incubated at 26 C. I took 1ml samples at the following time points: 0h, 1h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, 20h and 22h and then ran a gel and performed a Western blot.

Now that I have some results (well it looks like something to me ), I don't know how to interpret it.....

Please could someone have a look it for me? I have attached a scan of it above.

Thank you very much for any suggestions!

Sara

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Sara, could you please tell us what YOU think? you must at least have some idea, right?

it is very helpful also to label your standards, and to tell us the size of your protein of interest.

ps, what are these samples? pure extracts? column flow-through? we really need a bit more information

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Hi Sara --

Also, could you tell us a few other things? What vector your trans-sialidase was in? What was the final concentration of IPTG used for induction? What wavelength was used to record the OD of 0.6? Did you run a parallel uninduced culture as well, as a control? Did you look a Comassie stained gel (your Ab might not be recognizing your induced protein, but it will show up with Comassie)?

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Hi there,

Well I think, that some protein expression has occurred but I think there is also protein degradation. But what surprises me is that the lower bands are larger....I thought that if your protein (Which BTW has a His-tag) was "cut", aren't there supposed to be smaller fragments instead of larger ones??



The size of my protein of interest is supposed to be 75 KDa.....



The samples are from crude extracts

Thanks

Sara

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Hi Homebrew!!!

It's been a long time! Hey, I got my MSc in Bioinformatics (at last!).... and now doing my PhD and having such a hard time with these experiments

The vector was pTrcHis and the final conc of IPTG was 1mM. The wavelength I used was 600 and no I didn't run an uninduced culture

What is Comassie???

I think I should have stuck to Bioinformatics..... (at least I knew what I was doing)

Thanks Homebrew!!

Sara

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First things first -- congrats on the Bioinformatics MSc!

Now, to your experiment -- aimikins was asking you to describe the ladder you used because without us knowing the band sizes on the ladder, we can't know what band in your experimental lanes is ~75 kDa.

I don't see a whole lot of time-dependant over-expression happening on your western, but it's difficult to say. This is why, when I do these experiments (actually, I'm doing one know, coincidentally), I'll start with a culture twice as large as I need, and when it reaches the proper OD, split the culture in two, and add IPTG to only one. Then, I'll take time points from each culture during the induction phase. When I run the gel, I'll run uninduced and induced time points in pairs, so it's easy to see the induction.

I screen by staining the gel with Coomassie (a general protein stain -- see here) to assess the sucess of the induction, and -- if it looks good -- go to column enrichment of the tagged protein. Staining the gel aso is quicker -- instead of transfering the gel to a membrane, blocking it, incubating with primary and secondary Abs, and developing it -- not to mention all those washes in between -- you just dump the gel in the stain for a while, then dump it in destain for a while.

BTW, if you're going to screen with an antibody, you should also run a lane of host strain + vector only (no insert) lysate, to check for antibody cross reactivity with host- or vector-derived proteins.

The other thing that's helpful about using a general protein stain is that the concentration of all proteins will increase over time, as the culture grows. What you're looking for is for your protein to increase in concentration at a rate significantly higher than that of the other host proteins. With an antibody, you loose the "what is the concentration of irrelevant host proteins at this time point" reference, because you can't see them. The antibody will certainly be useful after your enrich for the protein by the column, but in assessing induction, I find Coomassie more useful.

Notice I said "enrich for" and not "purify"? The biggest myth of his-tag protocols is that they are a purification procedure. I've done hundreds of them, and have yet to see the end result be pure target protein -- there is always some host protein carry over. Thus, a general protein stain like Commassie is also needed to check your column fractions -- using an antibody will show you where your protein is, but not what host derived carry over also might be there. His-tag protocols are a pretty good enrichment procedure, but are far from a purification scheme.

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