Western Blot Protocol
(GPCRs antibodies)
Buffers and Solutions:
SDS-PAGE sample buffer:
2x Sample Buffer
Volume
Material
3,55 ml
Demonized water
1,25 ml
0,5 M Tris-HCl, pH 6,8
2,5 ml
Glycerol
2,0 ml
10 % (w/v) SDS
0,2 ml
0,5 % (w/v) bromophenol blue
9,5 ml
Total Volume
Add 50 µL of β-Mercaptoethanol to 950 µL of sample buffer before used.
Mix the sample to the sample buffer at least 1:2 proportion and heat to a 95
o
C for 4 min.
SDS-PAGE Running Buffer:
10x Running Buffer
Weight
Material
30,3 g
Tris base
144,0 g
Glycin
10,0 g
SDS
Dissolve in demonized water and fell to 1000 ml. Store at 4 oC. In case
something precipitated warms up the buffer before use.
OBS: DO NOT CONFIR THE pH.
Proteimax Biotecnologia Ltda.
www.proteimaxnet.com.br
Via das Margaridas, 413
Cotia-SP
SDS-PAGE Transfer Buffer:
•
25mM Tris-Base
•
192mM Glycin
•
20% Methanol
•
pH 8,3
For 1 L of buffer mix 3,03 g of Tris-Base, 14,4 g of glycin and 200 mL of
methanol; Complete to 1L with demonized water.
OBS: DO NOT CONFIR THE pH.
Protocol:
The amount of protein that should be loaded to the gel varies with the
experiment; it can be 10 to 50ug per sample (we are testing the best
concentration). Mixes 2 quantities of sample buffer for 1 quantity of sample and
warms up for 5 min at 100 oC. Some proteins need the addiction of more SDS (until
4%) directing through the sample or in the running buffer (0,4%).
Poliacrilamid Gel (8%):
Upper (4%):
Reagent
Volume
Tris 0.5M, pH 6.8
2.5 mL
SDS 10%
100 uL
Acrilamida 40% / Bis 1%
1.0 mL
APS 10%
50 uL
TEMED
10uL
H2O
6.40 mL
TOTAL
10 mL
Para gel 5% usar 1.25ml de Acrilamida 40% / Bis 1% e H2O 6.09ml.
Proteimax Biotecnologia Ltda.
www.proteimaxnet.com.br
Via das Margaridas, 413
Cotia-SP
Lower (10%):
Reagents
Volume
Tris 1.5M, pH 8.8
2.5 mL
SDS 10%
200 uL
Acrilamida 40% / Bis 1%
2.5 mL
APS 10%
50 uL
TEMED
5uL
H2O
4.74 mL
TOTAL
10 mL
Para gel 8% usar 2.0 ml de acrilamida 40% / Bis 1%, H2O 5.2ml.
After putting the sample in the gel, run until the blue marker goes of the gel.
The GPCRs band should be in the middle of the gel, but it is important to run a
marker together to make sure of the result. Run 2ul of blue marker. 80V 200amp.
Transference:
The transfer should be for 40 min at 10 volts in the semi-dry equipment from
Bio-Rad. Or over night at 15 V / 90 mA in the humid equipment.
Blocked:
The Blocked can be done with PBS 1x + 1% Albumin or with 5% milk in
PBST0.1%, the blot should be Blocked for 4-6 hours shaking (slowly) at room
temperature.
Primary Antibody:
After Blocked the blot without washing put the membrane in PBS 1x Tween
0,1% with the anti-GPCR in a 1:5000 dilution (on testing) and incubate it "over
night" at 4 oC in the shaker.
Proteimax Biotecnologia Ltda.
www.proteimaxnet.com.br
Via das Margaridas, 413
Cotia-SP
First wash:
After the primary antibody incubation wash rapidly 3 times the blot with PBS
1x and them wash 3 times (10 min with PBS 1x + Tween 0.1%). Don’t leave any
milk solution in contact to membrane.
Secondary Antibody:
After the washes add the secondary antibody (Licor 680 nm anti-Rabbit)
concentration 1:20000 in PBS Tween 0,1%, always protecting from light.
Second wash:
After the secondary antibody incubation wash rapidly 3 times the blot with
PBS 1x and them wash 4 times (20 min with PBS 1x + Tween 0.1%), always
protecting from light. Don’t leave any milk solution in contact to membrane.
Developed:
Scan in to the Odyssey. If there still some dirtiness washes more 3 times
with PBS 1x + Tween 0.1%.
Proteimax Biotecnologia Ltda.
www.proteimaxnet.com.br
Via das Margaridas, 413
Cotia-SP