TITLE: 2. Gel run and transfer -Tricine
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Western Blot</span></div></div>

[STEPS]
?. [Buffers:]
Tricine SDSRunningBuffer- dilute from 10 X to 1X Composition                               10X/L100 mM Tris base              (121 g)100 mM Tricine                  (179 g)0.1% SDS                             (10g)pH 8.3The pH of the 1X solution is 8.3. Do not use acid or base to adjust pH (Novex, 2003)Stable 6 months at room temperature
?. [Buffers:]
Tris-GlycineTransferBuffer-(Novex, 2003) Composition                            10X/L12 mM Tris Base              (14.56gm)96 mM Glycine                 (72 g)pH 8.3(1X solution is pH 8.3, do not use acid or base to adjust)Mix well and adjust the volume to 1 L. Store at room temperature, stable for 6 months.
Different concentrations found from different protocols e.g. this one was suggested for BioRad’selectrophoresis transfer cells TowbinBuffer with SDS25 mM Tris (3.03 gm/L),192 mM Glycine (14.4g/L), 20% MeOH (200 ml), SDS (0.1 g/L)- do not use SDS for small protein
?. [Running gel (See Invitrogen quick reference Cat. No. A25977, publication part no. 100025990).]
If running only one gel- make sure the unused chamber does not contain the cassette clamp.
?. [Running gel (See Invitrogen quick reference Cat. No. A25977, publication part no. 100025990).]
Prepare buffer (dilute 10X Tricine SDS Running Buffer to 1X)
?. [Running gel (See Invitrogen quick reference Cat. No. A25977, publication part no. 100025990).]
Plan to load 10 ul and with 5 ug of protein (might vary based on the concentration of your protein of interest). Prepare 1X sample buffer for dilution of samples, if needed. Allocate 12 ul (sample in SB containing 0.5 ug/ul) to an Eppendorf tube, also prepare your standard (1-1.5 ul of SeeBluePlus2 + 1X Sample buffer). Heat at 85 C for 2 minutes.
?. [Running gel (See Invitrogen quick reference Cat. No. A25977, publication part no. 100025990).]
Prepare gels.Remove the comb, and rinse wells three times using 1X Running Buffer.Remove the white tape near the bottom of the gel cassettes and place the gels in the tank.Load 10ul of your sample and standard. Adjust gels as necessary.Fill up the tank with Running buffer and Run (125 V constant ~90 minutes).
?. [Protein Transfer (see mini blot module Cat. No. B1000)]
Cut selected transfer membrane and filter paper to the dimensions of the gel
?. [Protein Transfer (see mini blot module Cat. No. B1000)]
PVDF membrane: Pre-wet PVDF membrane for 30 seconds in methanol,  ethanol, or isopropanol. Briefly rinse in deionized water, then place in a shallow dish with 50 ml of 1X Transfer Buffer for several minutes.  Standard PVDF has an unacceptable background signal in the IR.  Handle the membranes with forceps at the very edges, and minimize contact with gloves. Damage to the membrane can show up on the image of the blot. Use only dishes that are clean and have not contained Coomassie blue– the instrument is very sensitive and will detect traces of blue dye on the blot.  The blue dye in the pre-stained markers will be detected, which is convenient.  The red marker in the set will not be detected.
?. [Protein Transfer (see mini blot module Cat. No. B1000)]
Nitrocellulose: Place the membrane directly into a shallow dish containing 50 ml of 1X Transfer Buffer for several minutes.
?. [Protein Transfer (see mini blot module Cat. No. B1000)]
Filter paper: Soak the filter paper briefly in 1X Transfer Buffer immediately prior to use.
?. [Protein Transfer (see mini blot module Cat. No. B1000)]
Gel: Use the gel immediately following the run.  Do not soak the gel in transfer buffer.
?. [Follow mini blot module protocol (20V- 50 minutes for PVDF membrane)]
After the transfer is complete, turn the power supply off, unplug the power leads, and remove the lidRemove the blot module from the chamber and empty the transfer buffer, open module assembly, disassemble the blot sandwich, and carefully remove the membrane with blotting tweezersLet the membrane dry on a filter paper (store or proceed to immunodetection)