GGS LIVE - Antibody Generation

Hello BioFreakers,


Today in the GGS LIVE section we will cover how to generate rabbit polyclonal antibody:)

Method: Established methodology for antibody generation is an essential tool to study cellular functions of the protein of interest.

About: Antibodies are generated by injection of the antigen into host animal (mouse, rat, rabbit etc) and subsequent isolation of antibodies from the animal serum  (for instance from rabbit serum).

What: Generation of antibody against protein X.

There are many ways to generate antibodies but in our study case we are going to cover a protocol for generation of rabbit polyclonal antibody [monoclonal (recognise single epitope), polyclonal (recognises multiple epitopes)]. Ok, so lets see what are the main steps and tools here:

1. Antigen - design and generation - may be performed by you or company.
2. Animals - injection and animals maintanance - usually company
3. Validation of antibody - isolation and purification of antigen specific antibodies - you or company

In our study case, we will generate a polyclonal antibody against the protein of interest (size 25kDa). Because our protein is relatively small we can use its full sequence for rabbit immungenisation. In case of larger proteins (they still could be used as a whole, however purification of a big protein may be a limiting factor) a small peptide, usually from either N- or C-terminus of the protein is selected as an antigen. Please have a look at this animation showing an immune system response to antigens (Biology Animation -The Activatioof the Humoral and Cell-Mediated Pathways). This animation shows how antibodies are produced by the immune system.

Antigen generation

First we need to clone the Protein X DNA sequence into an appropriate vector that will allow us to express and purify the Protein X. In our study case we will use a GST tag for that purpose and we are going to purify it from E.coli cells. Cloning, expression and purification of tagged proteins have been previously described within different posts (just click GGS LIVE - PCR, -Cloning, -Protein tagging, -Expression and -Purification). Once we have the antigen purified (please see below):

we can contact the company that will generate the antibody for us. Before the immunogenisation we should check several pre-immune sera (serum from rabbits before the immunogenisation) to see if they are able to react with the proteins of a similar size as our protein of interest (important when antibody is going to be used primarily for immunoblotting aka Western blotting). Such a test by immunoblotting is shown below (click here for GGS LIVE - Immunoblotting tutorial):

As you can see, in our study case the test with pre-immune sera showed that all four sera react with proteins in lysates from human cells and detect a band of 75kDa. Moreover, three of them strongly recognise a band of molecular size of approximately 50kDa (sera 1, 3 and 4) and serum 2 picks up an extra band around 27kDa. Additionally, sera 3 and 4 recognise weakly two additional bands (size ~20kDa and ~30kDa for serum 3,  ~18kDa and ~35kDa for serum 4). This analysis allow us to pick up a serum (which corresponds to an animal that will be injected with the antigen) that does not recognise a band of a similar size as our protein of interest before immunogenisation. As you can see sera 1 and 4 are the only ones that do not pick up any bands in the area where our protein of interest may be migrating in the gel (between 20 and 35 kDa). Usually, the same antigen is injected into two animals to increase the chance of generating a working antibody, therefore we are going to choose rabbit 1 and 4 for our immunigenisation protocol.

After antigen generation and choosing the appropriate animals for immunogenisation, we send our antigen to the company which is going to inject it into rabbits and then provide us with immunogenised sera. This usually takes 4-12 weeks depending on the protocol used. During this time company usually provides three sera (1st, 2nd and final). These are then tested to see if they recognise the protein of interest using immunoblotting or any other immuno-technique (such as immunofluorescence or ELISA). Purified protein of interest (antigen - in our case Protein X) and cell lysates are used for these purposes. In the ideal situation the final serum (or even better 1st or 2nd one) recognises the protein of interest in the cell lysates without prior purification of the antigen specific antibodies from the serum. However, commonly the final sera recognises the antigen (a very good start and a must do in this case:)) but fail to recognise the protein of interest in the cell lysates. It also happens that the final serum is "dirty" and the immunoblots have a lot of non-specific, background signal that interferes with the detection of our protein (see below).

As you can see serum before and after the purification can easily detect the antigen but only the purified antibody can recognise the protein X in the cell lysates. This is very often due to the low concentration of the antigen specific antibodies in the serum compared to other antibodies. Therefore, the stronger the specific signal we want to obtain the stronger the background. This way it is very hard to get a strong enough signal (nice band on the right hand film above) for the protein X in the cell lysates that would be visible over the background noise. However, as you can clearly see purification of the antibody solved the problem (see here GGS LIVE - Antibody purification against the antigen).

Next step is to test if the antibody can be used in other applications such as immunofluorescence (GGS LIVE - Immunofluorescence) and immunoprecipitation (GGS LIVE - Immunoprecipitation).

I hope you enjoyed.

GGS TEAM     

GGS LIVE - Bacterial Transformation

Welcome Biofreakers,

Today, GGS TEAM is happy to present  the bacterial tranformation technique.

Method: Transformation of E.coli cells with DNA plasmid.

About: This technique allows for introduction of foreign DNA into bacteria cells in order to for example propagate the DNA or express a protein of interest.

 What: Transformation of E.coli strain TOP10 with pEGFPN1 plasmid containing MCM2 cDNA (pEGFPN1-MCM2).

Transforming plasmid DNA into bacteria cells is fast and easy. Of course we need bacteria cells and for that purpose different E. coli strains (such as TOP10, DH5alpha or others) are usually used. These cells are previously prepared in order to receive the DNA in the process that makes bacteria cells "competent". Such competency is nothing more than making the bacterial cell wall transiently penetrable (pores in the bacterial cell wall). Such condition of bacterial "coatings" allows for the take up of the DNA. Ok, lets start then. On the Figure below you can see an outline of the bacterial transformation process.

First competent bacteria are prepared using variety of different methods. These cells are then snap frozen using liquid nitrogen and stored at -80C to -150C to preserve to competent state. Once the cells are needed they are placed onto ice to defrost. On ice the cells are still competent but their ability to uptake DNA decreases with time and with increasing the temperature. Next, cells are mixed with DNA, in our case the DNA is the pEGFPN1-MCM2 plasmid. If the DNA that is used for transformation is a pure plasmid we do not need to use a lot but if the DNA we are using is for example DNA ligation reaction, we should use as much as possible to increase a chance of getting our DNA into the cells. After addition of the DNA the bacteria-DNA mix is incubated on ice for 20-30min in order to create "bacteria-DNA complexes". Next, the reaction is transfered to 42C (usually a water bath or hot plate) and incubated for a short time such as 90s. This step called heat shock opens previously introduced pores (these appeared when cells were made competent) and allows up-taking DNA that was previously in the contact with bacterial cell wall.After the heat shock there is a time for cold shock at 4C to close the pores and traps the DNA inside bacterial cells. After the "shocks" cells are allowed to recover by addition of fresh media and incubation at 37C shaking for approximately two cell cycles which in case of E coli is approximately 40min. Cells are then seeded onto plates containing appropriate antibiotic, in our case it is the Kanamycin and plates are the placed in the 37C incubator; and incubated over night. The amount of the cells seeded also depends on the DNA that was transformed. As previously mentioned, if the DNA was a pure plasmid we can seed very little (approximately 5-10% of the transformation) but if the DNA came from the ligation reaction we can seed all the transformation to be sure we get colonies back Next day, the colonies appear on the plates. Single colony is then picked up and expanded as a culture. Such culture can be then used to isolate bigger quantities of the DNA which later can be used for other purposes.

I hope you enjoyed my come back:)


GGS TEAM

GGS LIVE - Protein expression in vertebrate cells

Hello Biofreakers,

I am back BAYBE!!

Today we will cover a topic about expression of protein in vertebrate cells.

Method: Expression of protein X in human osteosarcoma cells (U2OS).

About: Protein expression allows for either purification or study of its cellular function through for example its localisation etc.

What: Expression of RFP-tagged-(Red Fluorescent Protein)-Protein X in U2OS cells to study its localisation.


To express a protein of interest in vertebrate cells we need to have its cDNA sequence. To get the target cDNA we need to clone it from either cDNA library (optionally isolate the cDNA from such library) or from mRNA. When cDNA sequence is ready it is then cloned into a vector that allows expression of that cDNA in vertebrate cells (please see this post GGS LIVE - Making a fusion protein for more details on how to create fusion proteins). Different vectors are available for expression of cDNAs in vertebrate cells. When vector containing cDNA is generated it has to be transfected into cells. This can be achieved by electroporation, chemical reagent such as lipid based transporters and others.

In our study case the U2OS cells will were transfected with lipid based reagent (lipofectamine). In order to detect the protein of interest its expression was monitored by fluorescence microscopy (please see this post for more details GGS LIVE - Immunofluorescence) as shown on Figure below. 

 

As you can see on the Figure, not transfected cell show very little or no red fluorescence indicating that there is no RFP-Protein X expressed in these cells. On the contrary the cells transfected with the cDNA coding RFP-Protein X fusion show red fluorescence. Moreover, the red fluorescence co-localises with the blue signal that comes from DNA (DNA was stained with DAPI). This suggests that the RFP-Protein X can be mainly found in the nucleus. Therefore, Protein-X is very likely to be a nuclear protein (to test that we could use technique described here GGS LIVE - Cell Fractionation). To confirm that we indeed expressed the RFP-protein X in U2OS cells, protein extract were prepared from these cells after transfection and analysed by immunoblotting (for more details on this technique please see here GGS LIVE - Western Blotting).

As you can see on the Figure below, the immunoblotting showed that RFP-Proiten X fusion can be detected exclusively in the cells that were transfected with the DNA coding for RFP-Protein X (antibody against the RFP was used to detect RFP-Protein X fusion). Detection of Actin protein was used here as loading control showing that the lane NT contains proteins but indeed does not have Protein X.

The biggest issue when expressing the protein of interest in the vertebrate cells is the transfection efficiency. The highest transfection efficiencies are obtained when the specific viruses are used to deliver the DNA into host cells (usually more thatn 90%). Unfortunately, I do not have any experience with this approach, therefore it will not be discussed further here. However, to ensure that as much cells as possible express our protein of interest we can optimise our transfection conditions by testing different ratios of the transfection reagent to DNA. For an example, we can keep the amount of the DNA constant and change the volume of transfection reagent as shown in the table and figure below:

To show you how this was quantified please see the pictures below:

In this case I used Fusion of Green Fluorescent Protein (GFP) with Protein X as my marker. The DNA labelling with DAPI shows the total number of the cells. The number of the cells showing Green Fluorescence by the total number of the cells gives us the transfection efficiency. As you can see when increasing amount of transfection reagent was used the transfection efficiency rised.

Moreover, the protein that we wish to express may not contain tag or be fused to a different type of tag (eg. non-fluorecent tag) that allows for example, to purify and study the biochemistry of the Protein X or to identify other proteins bound to protein X. This can be achieved by immnuprecipitation of the Protein X and analysis of the protein complexes associated with Protein X (for immunoprecipitation thechnique go here Biochemistry Methods - Immunoprecipitation).
As you can see the there is many ways that we could use protein expression as a tool to answer our scientific questions.

I hope you enjoyed it:)

Cheers,

GGS TEAM

GGS LIVE - Site Directed Mutagenesis

Hello BioFreakers!!

Today in the GGS - LIVE section the Site Directed Mutagenesis technique. Wanna see how it is done? Lets roll then.

Method: Site Directed Mutagenesis (SDM).

About: Allows for introduction of point mutations in DNA and thus sometimes in protein sequence.

What: Introduction of STOP codon in order to generate C-terminal deletion in protein X.

         Site directed mutagenesis is performed on circular DNA substrate. In our study case, the cDNA of protein X will be inserted into GST vector. Cloning of the protein X was performed analogously to study case described in a different post (click here GGS LIVE - Makinga fusion protein).

         To mutate protein X cDNA sequence we will need a set of primers that will be used in PCR reaction (GGS LIVE - Polymerase chain reaction (PCR)). SDM primers overlap with the target sequence, whereas regular PCR primers flank the target sequence (see cartoon below).

          The idea behind SDM is that each primer will mutate a single DNA strand of the pPLAS + X cDNA plasmid giving a product of the mutated pPLAS + X cDNA. Naturally, the template will be also present in the final mixture. Tamplate is removed by DpnI, a restriction endonuclease that digest modified DNA (DNA methylation). Such modification of the DNA occurs within bacterial cells, therefore SDM product will not wear it, as it was formed in vitro. After PCR reaction and overnight digestion with DpnI, SDM  reaction is analysed by agarose gel electrophoresis (see figure below).

         As you can see from the gel electroporesis analysis, desired product is present in both DpnI treated and untreated samples (R and R+D). Template +/- DpnI was used as a negative control. Additionally, amplification of 0.3kb fragment served as template control.
         You probably, wonder why SDM product and template are not observed as single band. This is due to different conformations of these plasmids. Template is supercoiled, therefore more packed and travels through gel faster and thus appears smaller on the gel. SDM product is not and therefore migrates slower.

         The R+D sample is then used to transform bacterial cells. In this process specific E.coli cells (for example Top10) uptake R+D plasmid and replicate it. This allows obtaining workable amounts of DNA, which is then send for sequencing. Several different E.coli clones are tested and when sequencing confirms mutation of pPLAS + X cDNA, DNA from correct clone is used to transform yet another specifc E.coli strain (for example BL21 pLysS).
          We then express X and X-truncation proteins using these BL21 cells (see figure below). For the protein expression in E.coli tutorial, please go here GGS LIVE - Protein expression in E.coli.

As you can see, this way we can generate a mutant protein KA-CHING. 

I hope you enjoyed it.

Maciek

GGSTEAM

GGS LIVE - Protein purification from E.coli

Yo Yo Biofreakers,

Today we are going to have closer look at the recombinant protein purification from bacterial cells.

Method: Purification of recombinant protein.

About: Having an optimised protocol for protein  purification is an essential tool to study properties of the protein of interest.

What: Purification of GST-tagged chicken protein previously expressed in E.coli.

Before we start we should first have a look at what the GST tag is. GST (glutathione S-transferase) is an enzyme that transfer glutathione (GSH) via slufhydryl group to differernt type of substrates (lipids, xenobiotics). GST has a high affinity towards its substrate glutathione and this property of GST is utilised to purify GST-tagged proteins. In order to recover GST-tagged protein from the complicated mixture of proteins, the fusion protein is incubated with agarose beads coupled to glutathione (GSH-agarose, see picture below), what leads to efficient precipitation of GST-fusion protein.

Ok Vamos!! Expression of the GST-X protein was previously demonstrated in different post GGS LIVE - Protein Expression in E.coli.


After a succesfull expression of the protein of interest in E.coli, we can purify it using one of the many protocols available for GST protein fusion purification. In general such protocol consists of three major steps: cell lysis and solubilisation of the GST-fusion protein (see cartoon below), recovery of the fusion from the lysate and elution of the GST fusion.

As you can see on the cartoon above, first GST-X fusion is expressed in large amount (for example 250 ml to 1 l culture) under previously optimised conditions (for protein expression see post GGS LIVE - Protein expression in E.coli). After protein expression, E.coli cells are harvested by centrifugation, superatant is removed and cell pellet is resuspended in lysis buffer of choice. Usually such buffer should have pH of around 7.0 - 8.0 to facilitate efficient interaction between GST and its substrate glutathione, protease inhibitors (such as PMSF) to prevent protein degradation. Additionally, lysis buffer should contain component taht will help lyse the cells, such as lysosyme (enzyme that degrades bacterial cell wall) or detergent (which disrupts bacterial cell wall). Cells are usually lysed at 4C rocking or mildly shaking what increases lysis efficiency. Cell lysate is then sonicated to share bacterial DNA (DNA makes lysate viscous and hard to work with) and help to solubilise proteins by breaking up protein aggregates. In the next step, cell debri is removed by high speed centrifugation. And there we go we have a lysate ready for protein purification.

As mentioned earlier, in order to recover our GST-X protein we have to mix our lysate containg the fusion with glutathione agarose beads. First beads have to be prepared (see cartoon below).

Glutathione agarose beads are first washed with the lysis buffer in order to remove storage solution (usually ethanol, which can impede binding of GST to glutathione). Then beads are mixed with lysate containing GST-X fusion and incubated at 4C in order to bind GST fusion to the beads. After binding step, beads have to be washed in order to remove unbound GST-X fusion and unspecifically bound proteins.

At this stage of purification GST-X fusion should be clean and depending on the nature of furhter experiments that we want to perform, we can either elute the fusion with glutathione (excess of the glutathione will compete and displace GST-X protein from the beads) or cleave the GST tag and release protein X (see cartoon below).

When purification is finished  we can analyse our experiment by separating protein sample taken at each step of the purification by SDS-PAGE and stain proteins in gel with Coomasie dye. The results from GST-X purification are shown on the picture below.

As you can see from the Coomasie stained gel, the expression of GST-X fusion was nicely induced (UI and I samples). We can also confirm that the GST-X was present in the starting material (lysate IN sample). After incubation of the lysate with beads, most of the GST-X bound to the resin what resulted in depletion of GST-X, as observed in unbound sample (UN). After beads wash, a single band of GST-X was detected on the beads, indicating high purifty of this sample. Elution of the GST-X with glutathione recovered fusion protein from beads. Additionally, alternative elution by GST cleavage resulted in appeareance of two bands: a free X protein and GST tag.

Hopefully, you got the picture how protein purification can be performed using a GST tag as a bait.

I hope u enjoyed it.

Cu SOON!

Maciek

GGSTEAM