Analysis of Proteins

This week and next, we will perform SDS-polyacrylamide gel electrophoresis followed by western immunoblotting on some of the remaining detergent extracts of transfected tumor cells. The goal is to confirm the presence of the appropriately-sized GFP fusion protein in the transfected cell lysates. For this you may choose any two of your ‘gene’ lysate samples- obviously you want to avoid choosing a sample for which there was a very low luciferase reading, as it might indicate that transfection efficiency was low for that particular sample. Also, choose one “vector” lysate sample. We are just choosing ‘representative’ samples for the western blot; we don’t expect the reactivity to be qualitatively different between the different gene samples or between the two vector lysate samples.

After successful ‘western’ transfer of proteins from the gel to a support membrane (PVDF paper), the membranes will be probed with specific antibody to the GFP ‘tag’; this process will be completed next week.

Background on SDS-polyacrylamide gel electrophoresis:

Polyacrylamide (vs. agarose) is the gel matrix of choice for fractionating most proteins, particularly those in the size range of our fusion proteins (~50-80 kDa). Electrophoresis is the process by which charged molecules are separated in an electric field according to their differing mobilities. Mobility, in turn, is affected by the net charge of the molecules, the voltage gradient of the electric field, and the frictional resistance of the supporting medium, which impedes movement of the ionic species. As a result of frictional resistance, larger molecules will be impeded to a greater degree than smaller molecules, and will have a decreased migration rate.

Polymerized acrylamide (polyacrylamide), like agarose, forms a porous gel matrix with sieving (separating) properties. The finer resolution allowed for by acrylamide vs. agarose is valuable for estimating the sizes of proteins and their subunits, which are generally much smaller in size than ds DNA fragments.

Importantly, the gel matrix also maintains the separation of different proteins in a mixture at the end of the electrophoresis run.

At a given pH, proteins have a net electric charge that is determined by their unique amino acid composition. When placed in an electric field, unmodified proteins will migrate toward the pole with the corresponding opposite charge. Most (but not all) proteins will have a net negative charge at a pH of 8 or higher, and will consequently migrate towards the anode (positive electrode) in an electric field. However, the rate of migration of different proteins under these conditions would be affected by both their charge and size, such that a larger protein with a high density of negative charge could migrate faster than (or equal to) a smaller protein with a lower density of negative charge.

To simplify the analysis of separated protein mixtures and make the separation strictly based on size of the proteins/polypeptide chains (in daltons or kilodaltons), the denaturing anionic detergent sodium dodecyl sulfate (SDS) can be added to the proteins. Like all detergents, SDS [(CH3 (CH2)11 OSO-3Na+] contains a polar “head” and a hydrocarbon (nonpolar) “tail”. SDS is commonly used to completely denature (unfold) proteins in preparation for electrophoresis, and coat them with a high density of negative charge that overcomes the intrinsic charge of the protein. The hydrocarbon tail of SDS binds to hydrophobic regions of amino acids, while the polar heads of SDS interact with polar residues of protein chains. Portions of the polypeptide backbone also participate in interactions with SDS. Noncovalent interactions that contribute to secondary, tertiary, and quarternary structure of proteins (eg., hydrogen bonds and hydrophobic interactions) are completely disrupted by SDS binding (in combination with heat), and the SDS-coated polypeptides have essentially an identical charge-to-mass ratio. In SDS-PAGE, the both running buffer and the protein ‘sample buffer’ contain SDS.

Unfolding- ‘denaturing’- the proteins is further aided by the presence of 2- mercaptoethanol (2-ME or β-ME) in the sample buffer, which is an optional component. This is a reducing agent that breaks covalent disulfide bonds that may contribute to the tertiary structure of a protein (eg., in extracellular proteins), or may hold multiple protein subunits together in a complex. SDS alone, even in the presence of heat, will not accomplish this. Unfolded, negatively charged proteins (SDS-coated) that are treated with a reducing agent such as â-ME will therefore migrate through the gel matrix toward the positive pole, at rates that are based solely on their sizes: i.e., larger proteins, impeded by the pores and frictional resistance of the gel matrix, move more slowly relative to smaller proteins. As a result, a protein mixture prepared in this manner that is applied to an acrylamide gel will form a ‘ladder’ based on size1, with the larger proteins nearer the top of the gel. This ladder of proteins can be visualized by staining the gel with appropriate dyes/stains, among other detection methods.

Acrylamide gels

The pores formed by copolymerization of acrylamide, a water-soluble monomer, with the cross-linking agent N, N’-methylene bisacrylamide (“Bis-acrylamide”) are of a size suitable for molecular sieving of most proteins and smaller DNA fragments. The sizes of the pores in acrylamide gels can be adjusted larger or smaller by changing the concentration of acrylamide monomer and/or bis-acrylamide cross-linker in the polymerization reaction. Gels with higher percentages of acrylamide- and smaller pores- are optimal for resolving smaller proteins, and v.v. Resolving gels can be cast with as little as 5% acrylamide or with up to 30%, but “typical” polyacrylamide gels are in the range of 7.5-15%.

In gels with a uniform concentration of acrylamide, there is a linear relationship between the migration distance of a protein (relative to an anionic dye at the migration front) and the log10 of its molecular weight. The linearity, however, may not hold at the extreme upper or lower end of the gel. In a typical 10% SDS-acrylamide gel (our gel of choice today), proteins from ~200 kDa (MW 200,000 daltons) to ~30 kDa (MW 30,000 daltons) will be resolved. In situations where a single gel must be used to separate and visualize proteins that differ widely in molecular mass, another option is to use a “gradient” gel in which the concentration of acrylamide increases linearly from the top to the bottom, for example a 4-15% gradient gel. The gradient in percentage of acrylamide creates a gradient in the degree of porosity such that the pore size at the top is relatively large (lower % acrylamide), and decreases towards the bottom of gel (higher % acrylamide). In this type of gel, the linear relationship is between the log10 of the proteins’ molecular mass and the log10 of the polyacrylamide concentration2 to which they migrate at the end of the run (another measure of distance). To cast this type of gel requires a specialized device called a “gradient maker”, which is simply a mixing/pouring device with separate chambers for the lowest and highest concentrations of acrylamide, attached to a peristaltic pump (leading to the gel plates).

SDS-PAGE and western immunoblotting of transfected GFP fusion proteins

Today’s procedures (in brief) will include:

1) running the polyacrylamide gels to fractionate the GFP fusion proteins (and other cellular proteins), followed immediately by

2) electrophoretic transfer of the proteins from the gel to a PVDF3 membrane (called a “blot” after transfer) for western immunoblotting.

3) Blot will then be placed into a covered plastic tray, submerged in ‘blocking buffer’, and rocked in the 40C cold room till next week. Blot will then be ready for probing with specific antibody to GFP.

Reagents, etc.:

• Subcellular lysates of transfected cells (prepared last week)
• Electrophoresis running buffer (10X stock, to be diluted down to 1X with dH2O)
• 4X reducing sample buffer (i.e., containing â- mercaptoethanol)
• Pre-stained protein molecular weight markers (‘standards’, labeled as “stds”); (analogous to the DNA markers); ready-to-load
• 10% acrylamide gels
• Transfer buffer (10X stock, to be diluted down to 1X with dH2O; see additional instructions below)
• Plastic tubs
• Reagents for ‘blocking buffer’, see below

Lab Instrumentation:

• Microcentrifuge
• Heat block
• Mini-gel apparatus, its power supply
• Transfer apparatus, its power supply
• Rocker

PVDF = Polyvinylidene fluoride, a hydrophobic support membrane with high protein binding capabilities and sufficient strength to withstand the handling associated with antibody probing (and re-probing).

Protocols:

Preparing the lysate samples for electrophoresis

1. Each team will have 3 lysate samples to run on the gel (aside from the molecular weight standards); one ‘vector’ lysate plus 2 of your ‘gene’ lysates (any two, as long as there was indication of successful transfection). Label a new eppendorf tube for each sample. To each tube that will contain ‘gene’ lysate, add 6µl of the 4X reducing sample buffer4 plus 18µl of the corresponding lysate from the procedure of last week. Vortex and pulse spin. (Note that you have diluted the 4X sample buffer down to 1X.) For the vector lysate sample, we usually need much less: prepare 2µl of 4X SB plus 6µl of vector lysate in a new tube. Vortex and pulse spin.

2. Obtain pre-prepared protein molecular weight markers (already in sample buffer, ready to load).

3. Heat all the samples EXCEPT FOR THE PROTEIN MOLECULAR WEIGHT MARKERS for ~3 minutes at 95ºC in the heating block (back bench of 406). This completes the denaturation of the proteins.

4. After heating, pulse-spin the tubes in the microcentrifuge to bring down the condensation from the top of the tube.

Preparing Running Buffer

Two teams will share a single gel (divided into ‘halves’ by a lane of molecular weight standards); one gel ‘apparatus’ (gel box) can hold 2 (or 4) gels. Therefore, we will need to run 4 gels total (for 8 teams). For convenience, we will probably divide them between 2 gel boxes (2 each) so as not to pressure all 8 teams to be ‘ready’ at the same time.

Each gel apparatus needs 700 ml of 1X SDS-Running Buffer (if running 2 gels), made from the prepared 10X stock (dilute with dH2O as usual). Mix by inverting in a parafilm-covered 1L cylinder.

Setting up the gel apparatus and running the gel

A demonstration of how to set up an acrylamide gel apparatus will be done. (The instructions below probably won’t make sense until you have seen the components of the system, so just use them as a guide after the demo.)

4X SB = A ‘concentrate’ of Tris-HCl, SDS, glycerol, pH 6.8, plus Bromphenol Blue tracking dye.

1. Insert the gels into the gel apparatus with the shorter plates facing IN towards the middle.

2. After 2 gels are loaded into a single module, fill the ‘inner’ chamber with buffer. “Rinse” each well 2 or 3 times with buffer from the inner chamber as demonstrated. Then load gel:

3. For each gel: Team #1: Pipette 10µl of the molecular weight markers (“stds”) into the left-most well. (Using the gel-loading pipette tips, you will load your wells by placing the pipette tip in between the 2 gel plates and into the well, without poking through.)

4. (Team #1) Then carefully pipette all 8µl of the vector sample into the next lane. In each subsequent lane, pipette in all 24µl of the prepared ‘gene’ lysate samples. Be sure to write in your lab notebooks which sample was loaded in each lane.

5. The next lane will be a ‘blank’ lane: load 10µl of 1X SB.

6. Team #2: Load 10µl of the molecular weight markers into the next lane.

Then, as in step #4 above, load your lysate samples into subsequent lanes. The final lane is another ‘blank’ lane, so load 10µl of 1X SB into this lane.

7. Attach the electrodes and run the gel at ~120-150 V until we have determined that sufficient separation of proteins has taken place. This will probably take ~30 min, but we will be looking for the bromophenol blue dye- front to be near the bottom of the gel, or at least in the bottom third of the gel.

8. For each gel: towards the end of the gel run, prepare 400 ml of 1X Transfer Buffer from the 10X stock, for the next step. Then add enough 10% SDS to obtain 0.01% SDS in the final 1X transfer buffer. Mix well in a cylinder and keep covered tightly with parafilm until needed.

Check on the gel periodically to be sure that you have current running through (you should see bubbles rising from the bottom wire). You should be able to see your blue dye front (bromphenol blue) migrating slowly through the gel, and the molecular weight markers should separate into distinct colored bands. Do not proceed unless you have a clear separation of molecular weight markers.

9. When the gel has finished running, remove the inner section from the gel apparatus as demonstrated. Slide down the green levers and remove your gel from its side of the holder; free the gel from its case by carefully prying the plastic plates apart with a spatula. The gel will stick to one plate initially. Place the gel (minus the plates) in ~150 ml of the 1X Transfer Buffer in a plastic tub for at least 15 minutes with gentle rocking. This step goes more smoothly if the gel and your gloves are kept wet. In a separate plastic tub, soak 2 pieces of thick filter paper and a precut piece of PVDF membrane (approximately 7 x 8.5 cm), pre-wetted with methanol*, in the remainder of the transfer buffer, also for ~15 min. The PVDF paper should be added to the buffer slowly.

* to save time, we will have pre-cut and pre-wetted (with methanol) PVDF paper for each gel; all you have to do is move the PVDF paper from water to transfer buffer

Transferring proteins to PVDF paper (immediately after electrophoresis)

Reagents:

• PVDF paper (= membrane= blot; cut to the same dimensions as gel)
• Acrylamide gel
• 2 thick filter papers (precut)
• 1X Transfer buffer
• Clean blotting containers (plastic tubs)
• Blocking solution (5% nonfat dry milk in 1X TBS-T)

Protocol: (Setting up Western Blot Transfer)

In the steps below, wear gloves when handling the gel and PVDF membrane.

1. Each gel requires one piece of PVDF paper; label with team names (in ink) on the bottom right half of the gel. Write as small as possible!

On the transfer apparatus, assemble the wetted papers and gel in this sequence (from bottom to top): Anode (plate electrode, see diagram), 1 piece of thick filter paper, your labeled PVDF, your gel5, second piece of thick filter paper, cathode-top plate). Keep in mind that if the PVDF is not on the correct side of the gel, the proteins will be transferred AWAY from the PVDF and lost. The transfer will run for 30 minutes at 15 V. Four gels can be run together on one semi-dry apparatus, but we may run 2 semi-dry units with 2 gels each, depending on the timing.

After placing the gel on the PVDF paper, use the roller device (as demonstrated) to gently roll out any air bubbles between gel and PVDF; repeat after the final piece of thick filter paper is placed on top. Tight contact is necessary for optimal transfer.

Semi-dry transfer apparatus

9. During the 30 minute transfer period, prepare blocking buffer for each gel/blot (see below).

Blocking solution

Although the antibody-antigen reaction we will use to detect GFP (next week) is highly specific, there is always some non-specific sticking of proteins to one another and to the PVDF membrane. To minimize non-specific protein binding on the blot, a protein-containing “blocking solution” (5% milk in 1X TBST) will be prepared and incubated with the blot initially. The milk proteins in the solution will bind weakly to the PVDF and will therefore prevent, or minimize, non-specific interactions with the antibodies (which are added afterwards). Prepare Blocking Buffer (easy!): 1X TBST/ 5.0% milk almost immediately, although we’ll keep it rocking if it’s made ahead of time. Blocking buffer is now ready to use.

TBST = Tris Buffered Saline with Tween-206 (a detergent)

1X TBST = 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.06%Tween-20

• For each gel/blot: you will need to prepare 10 ml of 1X TBST containing 5% (w/v) dry milk. A carboy of 2X TBST is available from which to make your 1X solution (dilute with dH2O); you may use a clean 15-ml blue-top tube for making the buffer. Calculate the amount of dry milk powder needed to constitute 5%.
•  First, prepare the 1X TBST and mix by inverting the tube. Then add the required amount of dry milk (weighed out on the balance in 404 or 406). Gently mix by inverting the tube several times. The milk should dissolve

Tween-20 is a detergent that helps reduce non-specific interactions of antibody with the membrane and with other proteins.

• After the transfer period is over, disassemble the semi-dry apparatus and discard the thick filter paper and gel. Place the PVDF blot (now stained with the pre-colored MW markers) in a tray of Milli-Q water for a brief rinse, then place blot in your prepared blocking solution in a smaller covered tray. The blot, submerged in blocking buffer, will be placed in the 4C cold room (on rocker) for one week, until the next lab period.

Questions:

• Immediately after transfer, how can you tell if you had a successful transfer of proteins from the gel to the membrane blot?

• Unlike DNA, in which all nucleotides have the same charge to mass ratio, amino acids/proteins have different net charges. How do we ensure that we are separating our proteins according to their size (MW, in kilodaltons) and not by their intrinsic charges?

Recipes for SDS-PAGE and Western Blot Analysis

10X Running Buffer
30 g Tris base (250 mM)
144 g glycine (1.92 M)
5 g SDS (0.5%) QS to 1 L

————-

2X TBS-T (Tris-buffered saline with Tween)
80 ml 1M Tris-Cl, pH 8.0 (40 mM Tris-Cl)
35 g NaCl (300 mM NaCl)
2.5 ml Tween-20 (0.125% Tween-20) QS to 2 L

————-

10X Transfer Buffer
250 mM Tris
1.92 M glycine
(optional: add SDS to 0.01%) (we will opt to add this) (pH ~8.5)