Using a Western blot, we will evaluate at what level our lab stock of HEK293 cells express Syk relative to the amount of Syk we plan to introduce as part of our BCR Tango system.

Knowing whether or not our HEK293 cells express Syk will better enable us to determine what level of Syk expression we need in our HEK293 model system and how much cross-talk we can expect from recruitment of endogenous Syk to the BCR upon antigen binding.

DNA Constructs:

pEXPR TRE:Syk-TEVp

Other:

Antibodies

Transfection

Gels:

We have the any kD gels in the lab (expire 2015) and this will work for both our western blot and antibody group's western blot.

If we run out of the above gel we are considering getting the 4-25% gel in order to optimize our resolution. This will also work for both our western blot and antibody group's western blot.

The gels currently in the lab have 10 lanes.  Here is the plan for the gel:

Since Syk-TEVp should run at a different band size than endogenous Syk, we should be able to identify the presence of both or either on a Western blot using anti-Syk antibodies. (There should be two bands, one corresponding to Syk-TEVp and the other to endogenous Syk.) We can use GAPDH (which we assume is produced at the same level in all of the cell populations we are testing) to normalize our data. This way, we can make comparisons between different cell populations while controlling for the amount of protein that we loaded in each lane. After washing the paper blot with anti-Syk and anti-GADPH, we should get three bands based on the sizes of the proteins:

GFP is added to control for variations in transfection efficiency for each transfection in the dox ladder. From the intensity of these bands, we should be able to determine the relative levels of endogenous and exogenous Syk expression.

Lysis buffer (from Abcam)

pH 8.0

150 mM NaCl

10% NP40 or Triton X-100

50 mM Tris

Running buffer (from Bio-Rad)

pH 8.3

25 mM Tris

192 mM glycine

0.1% SDS

Gel stain (from Abcam)

0.3 M CuCl₂

Gel destaining buffer (from Abcam)

pH 8.0

0.25 M Tris

0.25 M EDTA

Transfer buffer (from Abcam)

48 mM Tris

39 mM glycine

0.04% SDS

20% methanol

Membrane stain (Ponceau Red) - stock (from Abcam)

Dilute 1:10 for washing the membrane

2% Ponceau S

30% trichloroacetic acid

30% sulfosalicylic acid

TBST (Antibody/BSA buffer) (from Abcam)

100 mL TBS 10x (pH 7.6)

• 24.23 g Trizma HCl
• 80.06 g NaCl
• to 1L with dH₂O

900 mL dH₂O

1 mL Tween20

Block in 5% BSA (5g BSA in 100 mL TBST)

wrong ladder used - highest band = 40kd

gel not run long enough to distinguish larger proteins

loading control bands didn't show up - possibly because the blocking buffer we used contained casein (protein in milk) and abcam warned that using milk as a blocking buffer greatly reduced signal from anti-gapdh

syk bands didn't show up - possibly because we ran the gel under non-reducing conditions