Protein detection

2.7.1. Silver staining

After separating proteins by SDS-PAGE, gels were incubated for 15 minutes in fixer solution (50 % acetone, 1.25 % TCA and 0.015 % formaldehyde). The fixer was then removed and the gel washed repeatedly for 5 minutes with dH2O, before incubation

with 50 % acetone for 5 minutes. This was followed by a 1 minute incubation with a 0.02 % (w/v) sodium thiosulphate enhancer solution before additional washing in dH2O. The gels were then stained for 8 minutes using a solution containing 0.25 %

(w/v) silver nitrate and 0.4 % formaldehyde and washed with dH2O before inbuation

in developer solution (0.2 mM sodium carbonate, 0.004 % (w/v) sodium thiosulphate and 0.015 % formaldehyde). Once the protein bands were sufficiently visible the staining process was stopped using 1 % acetic acid solution. The gel was finally washed in dH2O before imaging.

2.7.2. Coomassie staining

After SDS-PAGE gels were incubated with Instant Blue Coomassie stain for over 1 hour before removal and subsequent washing in dH2O before imaging.

2.7.3. Protein transfer to PVDF membranes

A semi-dry Western blotting apparatus (Sigma, UK) was used to transfer proteins from SDS-PAGE gels onto PVDF membranes. After wetting the electrodes of the blotter two sheets of Whatman paper soaked in the transfer buffer were placed on the positive electrode. A methanol soaked PVDF membrane was then layered on top of the Whatman papers, followed by the gel. Lastly, two more pieces of buffer soaked

Chapter 2: Materials and Methods

40 Whatman paper were placed over the gel before closing the blotter. A uniform Towbin transfer buffer was used (25 mM Tris, 192 mM glycine and 20% methanol) (Towbin et al., 1979), and proteins were transferred at 200 mA over ~ 2 hours or at 20 mA over-night.

2.7.4. Immunoblotting

Following transfer of the proteins to PVDF membrane these were blocked to reduce non-specific binding of antibodies using either 5% (w/v) dried milk powder or 2% (w/v) bovine serum albumin (BSA) (for anti-strep II) dissolved in PBS-T. The membranes were left in blocking solution normally overnight, but at least more than 1 hour. After this the membranes were rinsed in PBS-T before the addition of 20 ml of the relevant primary antibody, diluted as shown in Table 2.7.4, for 1 hour. For

strep II tagged proteins, the membranes were incubated in 20 ml of biotin blocking

buffer (at 1:10000 dilution) for 10 minutes before the addition of the anti-strep II HRP conjugate for 2 hours. For the other primary antibodies, following repeated washing in PBS-T over 1 hour, the corresponding secondary HRP conjugate antibody was applied to the membrane; this was then incubated for 1 hour. Finally all membranes were washed repeatedly in PBS-T over at least 1 hour. Detection of the proteins was achieved using the EZ-ECL detection kit and X-ray films were developed using an AGFA Curix 60 automatic developer, as per the manufacturer’s instructions in each case. Antibodies used are listed below in Table 2.7.4.

Antibody Dilution Source

Mouse anti-his (C-terminal) 3:20000 Invitrogen, USA Mouse anti-strep II HRP conjugate 1:10000 IBA, Germany

Rabbit anti-TatAy 1:10000 Jan Maarten van Dijl

(University of Groningen)

Anti-rabbit IgG HRP conjugate 1:10000 Promega, USA

Anti-mouse IgG HRP conjugate 1:10000 Promega, USA

Table 2.7.4. Antibodies in this investigation

List of all antibodies used in this work along with the working dilution and original source of acquisition.

Chapter 2: Materials and Methods

41 2.8. Electron microscopy gridding methods

2.8.1. Sample preparation for EM in negative stain

20 µl aliquots were taken from the relevant gel filtration elution fraction. These were stored at -20C prior to use. Any dilutions were performed using the standard gel filtration buffer solution (20 mM Trizma-HCl, 150 mM NaCl) with or without detergent where stated. All solutions were filtered by hand through a 0.2 µm filter before use in dilution or washing steps.

2.8.2. Grids and stains used

Agar Scientific (UK): Formvar/carbon coated copper grids (300 mesh); lacey carbon grids; Uranyl acetate 2% or 1% (w/v) in dH2O (passed through 0.22 µm filter

prior to use).

2.8.3. Glow discharge

Glow discharge was performed with an EMtech K100x unit, running at 3x10-1 mbar and 25ma for 15 seconds of negative discharge in air.

2.8.4. Touching drop gridding method

The standard gridding method is given here; any modifications to this are presented later in the text where relevant:

4 µl of sample was applied to a grid within 10 minutes of glow-discharging and incubated on the grid for 1 minute before washing by touching the sample to an 8 µl drop of GF buffer for ~ 10 seconds with agitation. The grid was then blotted before quickly touching to a second drop of buffer and repeating this process. Next the grid was washed for ~ 10 seconds with an 8 µl drop of stain before blotting and staining with a second drop for 20 seconds. The grid was carefully blotted a final time before being left to dry. Adapted from a previously published method (Rubinstein, 2007)

Chapter 2: Materials and Methods

42 using a Parafilm® drop support and Whatman® paper for blotting, grids were left to air dry.

2.8.5. Cryo-plunging

For cryo-imaging lacey carbon grids were used. These are grids with a large number of irregular sized holes present in the carbon film where an ultra-thin layer of vitreous ice will form in which the particles of interest are imbedded. ~7 µl of sample was applied to a lacey carbon grid for 1 minute before blotting both sides simultaneously for 5 seconds and plunging the grid into liquid ethane following standard cryo-plunging methodology. An in-house machined pneumatic plunge system was used to achieve this.