Why do we need to remove the pellet in protein Extraction?

Hi,

I am trying to perform a luciferase assay using Applied Biological Materials (abm) plasmids, but I am uncertain on how much plasmid DNA should I use. I already decided that I will plate the cells in 24-well plates, and I know the amount of mimic miR that I will co-transfect the plasmids with but I can't figure what is the best plasmid DNA (WT 3'UTR, mut 3'UTR and empty vector) quantity, per well, to use.

I've been reading (a lot) and most authors fail to mention the DNA quantity used and when they mention, there isn't a uniform quantity. I've already read reports in which authors used 50 ng/well while in others, used 1 ug/well.

If anyone already used this plasmids and knows how to proceed, I would be extremely grateful if you could help me.

Best regards,

Gonçalo Pinho

I'm currently working on my fourth try of transformation. Every time the plates have A LOT of tiny colonies (photo attached). Every once in a while there is a larger colony, but when I screen them, they don't contain the recombinant plasmid. The negative control has colonies also, but the plate doesn't have the tiny colonies covering it like the other plates.

I've redone the digestion and still get the same results. I checked to make sure the plates contain ampicillin by transforming bacteria with a plasmid resistant to kenomycin. No bacteria grew when I did this. I've tried troubleshooting, but there are a lot of things I can change, and I'm not sure where to start. Is the ampicillin concentration in the plates not high enough (I've been using 500 uL for 500 mL of agar), am I using too much of my ligation reaction (I've been using all 10 uL), am I plating too much (I've been plating about 350 uL)? Has anyone had this issue before?

Also, when you screen your colonies, what else do you run on the gel? I vaguely remember doing cloning in another lab and when we screened our colonies we ran the uncut vector and the cut vector, but I've only been running the negative and positive controls.

I extracted rice leaf (fresh leaf) total protein using following protein extraction buffer on ice bath or at Room temperature (without liquid-N2). After that, I did Western blot (WB) to check my gene expression. I saw band in X-ray film and gene expression is confirmed as my gene have FLAG-Tag epitope, it was easy for me to detect by using anti-flag tag antibodies ( I attached the antibodies catalog which are used by me, picture 1 and 2).

My protein extraction buffer (QB solution) is:

100mM KPO4 (pH 7.8)

1mM EDTA

1% Triton X-100

10% glycerol

1mM DTT (added just before use)

But when I DO NOT add DTT in my extraction buffer (QB solution except DTT), I cannot see any band in Western blot X-ray film for the same sample. Why it is happening, I do not understand.

** Many protein extraction buffers do not use the DTT or other reducing agent. After that they found their expected protein (confirmed by WB). What is the function of DTT during protein extraction?

*My ultimate goal is to purify protein by using affinity agarose resin column. But my agarose resin kit does not allow the use of DTT.

My agarose resin column (I attached the resin column catalog number, picture no 3) Kit:

Does not allow the use of: urea, guanidine HCl, DTT (Dithiothreitol), DTE (dithioerythritol), 2-Mercaptoethanol, SDS (sodium dodecyl sulfate)

Can use in less % : Tween 20 (up to 5%), Triton X-100 (up to 5%), NaCl (up to 1M)

So one time I changed my protein extraction buffer to (50mM Tris-HCl ph7.5, .5M NaCl, 1mM EDTA, 1% Triton X-100, 10% glycerol, 1% PVPP) and I did this on ice bath and also used liquid-N2 to grind leaf (fresh leaf). But I did not get any band in Western blot X-ray film.

***Protein extraction by QB solution: is problem with the use of DTT as DTT reduces disulfide bonds. So my resin column prohibits to use this kinds of reagents that reduces disulfide bonds (please check picture no. 4).

*** Can I extract protein without DTT (extraction buffer QB solution except DTT ) for purification of protein? But why I cannot see any band in WB X-ray film?

*** Is it the problem of not using DTT Or it is because of antibodies detection limitation? As I saw in datasheet of secondary antibody images, they did WB in reducing condition (please check picture 5)?

All these things, making me confused.

In this circumstances, how can I overcome these problem?

Any suggestion will be highly appreciated!

i centrifuged at 14000 rpm 20 min but not pellet formed. 

I am currently in the process of western blot and discovered that I have stored the protein at 4C...

The sample was harvested about 6days ago and mixed with SDS sample buffer then boiled for 8mins, then stored at 4C.

Will this affect my results...?

Thank you

Davis

Why does my protein stay in the pellet of the lysate?

I expressed a KPC-2 Protein in NP6 cells the vector is PqE-2. After lysis (8M urea in a tris buffer pH 8), the protein is in the pellet. I have tried many different things like  different IPTG concentration 0.1Mm,0.2,0.3,0.4 and 0.5mM. sonication, microfluidizer, triton x-100, lyzozyme, and SDS. But I am unable to obtain satisfactory result. What else can I do to bring my protein into solution?

I expressed a cystein protease in M15 cells the vector is pQE30-xa. After lysis (8M urea in a tris buffer pH 8), the protein is in the pellet. I have tried many different things like 6M guanidinium hcl, sonication, microfluidizer, triton x-100, lyzozyme, and SDS. The only one that worked was SDS but since I need to do enzymatic assays SDS will interfere. What else can I do to bring my protein into solution?

Hi everyone. Does anyone know what the sample final volume is for using HiTrap Protein A HP (1ml) antibody purification columns?

Thank you very much in advance