Western blotting (immunoblotting) to probe for GFP fusion proteins in transfected cell lysates

In today’s procedure, you will probe your blots with antibody to the GFP ‘tag’ on your fusion proteins; this will ultimately reveal a protein band whose molecular weight (MW) is a combination of the GFP protein’s MW (~26 kDa) plus that of the protein product of your gene. This value can be determined by counting the nucleotides in the ‘grey’ plus ‘green’ regions of your color-coded gene diagram. Begin “word count” with the first grey-shaded nucleotide, and count the entire area through and including the red “stop” TAA codon at the end of the ‘green’ GFP region. Divide this number by three to obtain the number of codons (which is the number of amino acids coded for); multiply this by 110* to obtain the approximate MW (in daltons) of your fusion protein. For ex., 1500 nucleotides means 500 codons and therefore 500 amino acids; 500 x 110 = 55,000 daltons (or 55 kilodaltons, kDa).

* The average molecular weight of an amino acid is 110 daltons

Overview of a typical immunoblot procedure:

A specific, single protein on a gel can be visualized through immunoblot analysis with an antibody that recognizes the protein of interest. To perform an immunoblot, the separated proteins within the gel are first transferred onto a membrane (usually nitrocellulose or PVDF) that can be treated with antibody solutions. After preliminary incubation of the membrane in excess ‘inert’ protein (eg, milk) to block non-specific protein binding reactions, the membrane is then incubated with a primary antibody, which is an antibody that was generated to specifically recognize the ‘protein of interest’. For our purposes this target protein is GFP, the common “C-terminal tag” of all the fusion proteins. The excess primary antibody is removed after a brief incubation period, then the blot is “washed” with buffer to remove non-specifically or weakly bound antibodies.

After this, a secondary antibody that recognizes the first (primary) antibody is incubated on the blot. This secondary antibody, which is raised against antibodies of a specific species, (e.g. anti-mouse antibodies or anti-rabbit antibodies) is covalently linked to an enzyme (typically horseradish peroxidase, HRP) that catalyzes a localized light-generating reaction in the presence of substrate. This is called chemiluminescent detection (using chemical reactions to generate light) and the emitted photons are captured using the CCD camera of the BioChemi imaging unit. The chemiluminescent substrate mixture for HRP consists of peroxide buffers and a proprietary variation of luminol designed to generate light from very small quantities of protein.

Ultimately, this produces an image of your blot with ‘white’ glowing bands on a black background. The image can be ‘reversed’ for clarity, and these images typically are, prior to presentation in a report or publication.

Antibodies as tools for detection of proteins:

Among all the proteins extracted from the transfected tumor cells and resolved in the electrophoresis step, the western blot allows the detection of a single protein, or more specifically, a single protein domain or epitope, on the blot.           For the detection of your GFP fusion proteins, we are using an antibody that binds specifically to GFP, and should therefore detect each fusion protein regardless of what the ‘fusion partner’ is.

In a western immunoblot, the primary antibody could be ‘monoclonal’ or ‘polyclonal’, and the choice made by the user is typically based on the most effective antibody available for the given protein and/or the specific information needed. A monoclonal antibody is derived from a single clone of the antibody- producing “B” cells of an immunized mouse or rat2. All of the antibody (Ab) molecules in these preparations have identical specificity in that they recognize the same epitope3 on the target protein. In contrast, a polyclonal antibody preparation is a combination of the antibody products of multiple “B” cell clones; these are typically derived from the sera of immunized rabbits, goats, or sheep, and they collectively recognize multiple epitopes (regions) on the target protein. Whether you use a monoclonal or polyclonal antibody as your primary antibody in a western blot depends on a number of things -availability, effectiveness, cost- but most importantly on whether you need the precise specificity that a monoclonal Ab provides (in binding to only a very specific region of the protein) or whether it would be sufficient for a ‘larger’ region to be recognized, in which case a polyclonal Ab would work just fine. Our antibody against GFP (anti-tGFP) happens to be a mouse monoclonal antibody, purchased through Origene.

The secondary antibody (an “anti-antibody”) in a western blot protocol is, by definition, isolated from a species other than the species that produced your primary Ab. This antibody (typically polyclonal) will bind to multiple common determinants (i.e., epitopes) on antibodies from the species that generated the primary antibody. The secondary antibody may be referred to as a “rabbit anti- mouse” antibody (derived from rabbit; antibody to mouse antibodies), a “goat anti-rabbit” antibody (derived from goat; antibody to rabbit antibodies), “sheep anti-rabbit antibody” (derived from sheep; antibody to rabbit antibodies), etc. Our secondary antibody for use in GFP detection is an HRP-linked goat anti-mouse antibody, or “GAM-HRP”, purchased from Pierce.

Immunoblot Protocol- adding primary antibody:

Two hours prior to lab, you will come to lab very briefly to add primary antibody to your blocked blots. Remember to keep track of which team is on which ‘half’ of blot.

1. First, pour off (in sink) the blocking solution from the PVDF blot.
2. To each tray, add ~8-10 ml of the prepared primary ‘anti-GFP’ antibody

*The primary antibody solution consists of: a mouse monoclonal antibody (mAb) that recognizes tGFP, diluted 1:20004 in 1X TBST with 0.5% milk. (Clone 2H8, cat# TA 150041, from OriGene Technologies Inc.)

3. Close the tray and place on the rocker at the back of 406. The blots will incubate in primary antibody solution at room temperature for ~2 hr.
4. The optimal dilution for a primary Ab in any western blot protocol is determined by the end user, and often involves trial and error of a range of dilutions until the one is found which gives the highest “signal” to “noise” ratio (with “noise” referring to background).

Washing, secondary antibody incubation, and development:

Each blot will require ~300 ml of 1X TBST (no milk), prepared from the 2X concentrate, for washing purposes; if more is needed, it can be made up quickly; leftover 1X can be saved.

At the end of the 10 antibody incubation period,

1. Pour off and discard primary antibody, taking care to support PVDF membrane with gloved finger(s).
2. Rinse the blot two times for a few seconds each, with enough 1X TBST to cover blot, pouring each rinse down the sink.
3. Perform three subsequent washes of the blot, each with sufficient 1X TBST to cover the blots (more than 10 ml, but not so much as to spill out of the tray). Wash by rocking the submerged blot in its closed tray, on the rocking platform as follows:

•      10 minutes5 (then discard the wash down the sink)

•      5 minutes (discard wash solution as above)

•      5 minutes (do not discard immediately; see step #4 below)

4. During the final wash (or before), 10 ml of secondary antibody solution needs to be prepared for each blot (Goat Anti-Mouse, HRP-conjugated). The GAM-HRP will have been diluted (by instructor) by 1:10,000 (a ‘substock’) in the same buffer as the primary antibody (1X TBST/0.5% milk); you need to further dilute this substock by 1:5, for a final dilution of 1:50,000. To do this, simply add 2 ml of the GAM-HRP substock to 8 ml of 1X TBST/0.5% milk (provided) in a 15 ml tube. Mix by inverting a few times.
5. Discard the final wash solution from step 3; add the entire 10 ml of the prepared GAM-HRP solution from step 4. (Do NOT let the blot dry out!)
6. Incubate on the rocking platform in 406 for ~45 minutes.
7. After the 45 min incubation, discard the secondary antibody solution down the sink, and repeat the wash sequence from Step #2 and Step #3. Do not discard the final wash solution; let the blot ‘sit’ in this solution, keeping it wet, until its final incubation in substrate solution, see below.
8. During the last wash, make up the “Femto” western blot substrate solution (working reagent) for each blot as follows: to a 15 ml blue-top tube, add 5 ml of each of the two provided solutions: Luminol (dark bottle) and Peroxide buffer. Mix by gently inverting the tube.
9. Using a separate small clean tray, first place the Femto solution into the tray. Using forceps, lift the blot from its final wash solution and briefly dab excess liquid onto a piece of Whatman paper. Place the blot ‘face down’ (protein side down) into the Femto solution. Incubate 5 minutes (no need to continuously shake, although do make sure the entire blot surface is being wetted).
10. After the incubation is complete, remove the blot from the solution, place ‘face up’ (protein side up) on dry Whatman filter paper, and blot dry with a second sheet of dry Whatman paper. (Instructor will demonstrate.) It is probably wise to wait until you are “next in line” for the BioChemi unit before doing the substrate (Femto) incubation with your blot. In the meantime, your blot can safely ‘wait’ in the tray with the final wash solution.
11. Instructors will assist you in obtaining an image of your blot using the

Insert Image

Biochemi imaging unit.

Protein size can be estimated by comparison to known molecular weight standards that are run beside the experimental samples. We have used BioRad’s “Kaleidoscope” as our molecular weight standards (see pdf in Bb for labeled image); these are recombinant pre-stained proteins designed to provide a highly accurate ladder of convenient sizes. [A standard curve generated by plotting the log of their molecular weights vs. migration distance (Rf) in a SDS- PAGE gel reveals a straight line with r2 = 0.996.]

Questions:

• • What was the expected size (in daltons or kilodaltons) of your fusion protein? What is the expected size of GFP ‘alone’, from vector lysates?
• • Did you get the results you expected?
• • If not, what are some possible explanations?
• • A primary antibody to GFP, covalently linked to HRP, would also provide a specific signal in our western blots. What are some of the advantages, then, of doing the antibody incubation in two steps (primary followed by secondary)? Why not just omit the secondary antibody step?