Syk Western Blot

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)

Results of BCA Protein Assay

A BCA Protein Assay was run to determine the concentration of protein in our samples. In the assay, protein samples were analyzed in two dilutions: 75uL sample + 75uL dH₂O and 25uL sample + 125 uL dH₂O. These were compared to BSA protein standards. 150uL of each dilution of sample or BSA was added to 150uL of BCA working reagent (25 A: 24B: 1C) in a microwell plate and scanned using the Tecan plate reader at 562nm absorbance. A standard curve was created, and absorbances of the samples were compared against this curve to produce predicted protein concentrations.

See attached document for details of experimental results. In this iteration of the Western blot, 10uL of undiluted protein sample was added to 10uL of sample buffer, and 16uL of this solution was added to each well. Based on the concentrations inferred from the BCA assay and our protocol for loading the gel wells, the calculated amount of protein added to each well was:

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

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.

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)

Results of BCA Protein Assay

A BCA Protein Assay was run to determine the concentration of protein in our samples. In the assay, protein samples were analyzed in two dilutions: 75uL sample + 75uL dH₂O and 25uL sample + 125 uL dH₂O. These were compared to BSA protein standards. 150uL of each dilution of sample or BSA was added to 150uL of BCA working reagent (25 A: 24B: 1C) in a microwell plate and scanned using the Tecan plate reader at 562nm absorbance. A standard curve was created, and absorbances of the samples were compared against this curve to produce predicted protein concentrations.

See attached document for details of experimental results. In this iteration of the Western blot, 10uL of undiluted protein sample was added to 10uL of sample buffer, and 16uL of this solution was added to each well.

The correct ladder was used this time, which enabled us to determine which bands belonged to which of the proteins we were probing. 5% BSA blocking resulted in stronger bands than Odyssey blocking; however each gave some nonspecific bands. As expected, all cells appeared to be expressing GAPDH, though the levels of GAPDH were barely detectable for the Ramos samples because of low starting protein concentration (see BCA assay). Based on the combined results from the Odyssey and BSA blocking, both Ramos and HEK293 appear to express Syk at detectable levels. As expected, Syk-TEVp was expressed at higher levels in samples with higher doxycycline concentrations. Based on a qualitative visual analysis of the bands, endogenous Syk expression appears to be at a similar level to Syk-TEVp with 2000nM dox induction. This indicates that we may have cross-talk issues or signal dilution in our HEK293 model relative to a cell model that has no endogenous Syk.

Future considerations:

• Continue to do SDS-PAGE rather than native PAGE. All of the bands showed up with SDS-PAGE, which was not true of native PAGE.
• Continue to use BSA blocking. It appears to give better signal than Odyssey blocking. Or try using a combination of both.
• Consider using a lower concentration of anti-GFP. The signal is really bright relative to the Syk and GAPDH signals.
• Identify the sources of nonspecific binding. Since it shows up at 700nm, it is probably anti-GFP (since this is also polyclonal). Longer incubation at a colder temperature or reducing the amount of anti-GFP added could potentially reduce nonspecific binding.
• Load more protein. Grow cell populations in larger wells (to get more cells per well) to get higher protein concentrations. This should give brighter bands, particularly for Syk, Syk-TEVp, and GAPDH.
• "Phantom" bands appeared in lanes adjacent to the ladders. Consider switching ladders to keep this from happening, since it didn't happen when we used the wrong ladder.
• Is 2000nM dox actually saturating the TRE promoter?