Western Blot Protocol for use with GPCR Polyclonal Antibodies from Proteimax

Date Published: July 18, 2023

Note: The following protocol has been developed for use with GPCR polyclonal antibodies manufactured by Proteimax. Other antibodies have not been tested using this protocol. For additional assistance, please contact Technical Support.

Buffers to prepare:

1.  2X SDS-PAGE Sample Buffer

Volume

Material

3.55 ml

Deionized 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 ß-Mercaptoethanol to 950 µL of sample buffer before use. Mix the sample to the sample buffer at least 1:2 proportion and heat to 95°C for 4 minutes.

2.  10X SDS-PAGE Running Buffer
Dissolve the following reagents in deionized water to a final volume of 1000 mL. Store at 4 °C. Warm the buffer before use to remove possible precipitates. Do not pH adjust.
Amount          Material                         
30.3 g Tris base
144 g Glycerin
10 g SDS
3.  SDS-PAGE Transfer Buffer (25 mM Tris-Base, 192 mM glycine, 20% methanol)
For one liter of buffer, mix 3.03 g of Tris-Base, 14.4 g of glycine and 200 mL of methanol. Add deionized water to one liter final volume. Do not pH adjust.
Western Blot Protocol:
The amount of protein that should be loaded to the gel varies with the experiment. It can be 25 to 100 µg per sample. For total brain membranes, 50 µg is recommended.
1.  Mix equal volumes of 2X SDS-PAGE Sample Buffer and sample, then heat for 5 minutes at 65°C.
2.  Load the sample in 8% polyacrylamide gel, together with marker. Refer to SDS-PAGE gels below:

  Upper Gel (8%)

  10 ml

  20 ml

  50 ml

  0.5 M Tris, pH 6.8

  2.5 mL

  5.0 mL

  12.5 mL

  SDS, 10%

  100 µL

  200 µL

  500 µL

  Acrylamide, 40% / Bis, 1%

  1.0 mL

  2.0 mL

  5.0 mL

  APS, 10%

  50 µL

  100 µL

  250 µL

  TEMED

  10 µL

  20 µL

  50 µL

  Water

  6.4 mL

  12.8 mL

  32 mL

  Lower Gel (8%)

  10 mL

  20 mL

  50 mL

  1.5 M Tris,  pH 8.8

  2.5 mL

  5.0 mL

  12.5 mL

  SDS, 10%

  200 µL

  400 µL

  1.0 mL

  Acrylamide, 40% / Bis, 1%

  2.0 mL

  4.0 mL

  10.0 mL

  APS, 10%

  50 µL

  100 µL

  250 µL

  TEMED

  5 µL

  10 µL

  25 µL

  Water

  5.6 mL

  11.2 mL

  28 mL
3.  After loading the sample, run gel until the blue marker goes to the end of the gel. The GPCRs band should be in the middle of the gel, but it is important to run a marker together. Gels are generally run at 15mA (constant current) until the samples are into the upper gel (approximately 15-20 minutes) at which time the current can be increased to 25-35mA until the dye front is just about to leave the gel (approximately 60-90 minutes).

Transfer:
The transfer should be semi-dry for 40 minutes at 10V or wet overnight at 15V/90mA.

Blocking:
Block with either 1X PBS + 3% Albumin or with 5% milk in PBS. Block for 4-6 hours at room temperature while slowly shaking.

Primary Antibody:
After blocking, without washing, put the membrane in 1X PBS with the anti-GPCR antibody in a 1:6000 dilution and incubate overnight at 4°C in the shaker. Note that the optimal dilution for a specific antibody will vary and should be determined by the end user. See the product-specific specification sheet for recommended starting dilutions.

First wash:
After the primary antibody incubation, wash the blot three times with 1X PBS and then wash three times (10 minutes with 1X PBS + 0.1% Tween).

Secondary Antibody:
Incubate for two hours in 1X PBS + 0.1% Tween with the secondary antibody (Licor 760 nm anti-Rabbit) at concentration 1:10,000 (or according to the manufacturer’s protocol).

Second wash:
After the secondary incubation, wash rapidly three times with 1X PBS + 0.1% Tween. Then wash three times, for 20 minutes each wash, with 1X PBS + 0.1% Tween.

Develop:
Before developing the blot, wash 3 times with PBS and scan in the Licor. If there is still some dirtiness, wash an additional three times with 1X PBS + 0.1% Tween.

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