Download Detecting hydrophobic proteins by western blot

Sam’s molecular biology protocols
Making Trypanosome protein samples for detecting
problem transmembrane proteins
Some hydrophobic proteins are not amenable to detection by standard western
blotting protocols. This has been shown to be due to a problem hydrophobic domain
in the protein (rather than due to its global hydrophobicity) that is not completely
denatured by SDS and boiling. When this problem domain is exposed it is not fully
denatured by the boiling and detergent and ultimately prevents the protein from
entering the acrylamide gel.
Ref:
Okada, N. et al. Identification of TMEM45B as a protein clearly showing thermal
aggregation in SDS-PAGE gels and dissection of its amino acid sequence
responsible for this aggregation. Protein Expr Purif 77, 118–123 (2011).
Lee, Y.-N. et al. Thermal aggregation of SARS-CoV membrane protein. J Virol
Methods 129, 152–161 (2005).
Notes
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At each stage it is important to remove as much of the supernatant as you can
in order to remove residual FCS.
Use protease inhibitors to avoid degradation of your sample during processing.
Note that the reducing agent in the Laemmli buffer should either be added
fresh, or once added the Laemmli buffer should be stored frozen.
You can substitute LDS for SDS in the Laemmli buffer (this is available
commercially), and keep everything on ice at all times.
In this case it may be necessary to shear the gDNA mechanically (by either
water bath sonication or using a needle)
When making protein samples from blood stream form cells, use vPBS in
place of PBS to keep them happy during processing
I load 3 x 106 cells per lane (=6l)
You will need:
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Ideally about 5 x 107 cells (enough for 16 Westerns)
4x Laemmli buffer with 0.1M DTT added (optional – swap SDS for LDS)
PBS (or vPBS for bsf cells)
Protease inhibitors
o Leupeptin hemisulfate salt (Sigma L2884), 20mM 400x
o Pepstatin A (Sigma P5318), 3mM 400x
o PMSF (phenylmethylsulfonyl fluoride) (Roche 10837091001), 200mM
400x
o E-64 d (Biomol/ENZO PI-107)], 5mM 1000x
Sam’s molecular biology protocols
Protein preparation
1. Count cells using Casycounter. Unless there is a good reason not to, use cells
in log phase
2. Pellet about 5 x 107 cells in a 15 or 50ml falcon, 800g for 10 minutes
3. Carefully pour off supernatant
a. Remove as much of the media as you can by gently touching the falcon
to some blue roll
4. Re-suspend the pellet in 1ml of PBS (or vPBS for bsf) containing 2x protease
inhibitors
5. Transfer cells to a 1.5ml eppendorf
6. Centrifuge at 1000g for 5 minutes
7. Remove the supernatant by aspiration
a. Remove as much of the PBS as you can without disturbing the pellet
8. Re-suspend in PBS (or vPBS for bsf) containing 2x protease inhibitors at a
concentration of 1 x 106 cells.l-1
a. If you started with 5 x 107 cells, this equates to 50l
9. Place cells on ice for a couple of minutes
10. Add an equal volume of cold 4x Laemmli buffer
a.  the final cell concentration is 0.5x106 cells.l-1
11. It may be necessary to shear the DNA to get it to enter the gel nicely
a. For larger sample volumes a needle is the best way
b. For smaller volumes, use an ice-water bath for 5-10 minutes (but
careful you don’t have water leak into your samples!!)
Running the gel PAGE gel
1. Use conventional PAGE gels and buffers (Tris-gly, or bis-Tris, containing
SDS)
2. Centrifuge your samples max speed in a chilled rotor
3. Run your samples at 100V in the cold:
a. Pre-chill the buffers overnight in the fridge
i. If you forget to pre-chill your buffers, incubate them in an icewater bath until they are cold
b. Perform in the cold room
c. Keep your tank in an ice waterbath
4. Start early, as it takes a while
Transferring proteins onto membrane
1. Perform a wet transfer onto PVDF or nitrocellulose, overnight at 15V in the
cold room.
2. Vary the methanol concentration according to the size of the protein (i.e.
larger proteins should use less methanol)