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Full Western Blot - TJ

Full Western Blot - TJ[]

Brief Description

General Western Blot Protocol - includes gel concentrations

Protocol Details

Western Blot Gel Set-up

SDS-PAGE GELS

 

Separating Gel

%

5

6

7

7.5

8

9

10

12

13

15

16.5

30% acrylamide

1.67

2

2.33

2.5

2.67

3

3.33

4

4.33

5

5.5

separating buffer

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

2.5

ddi H2O

5.83

5.5

5.17

5

4.83

4.5

4.17

3.5

3.17

2.5

2

10% APS 50uL

TEMED 12uL

 

Stacking Gel

30% acrylamide .67mL
stacking buffer 1.25mL
ddi H20 3mL

10% APS 30uL

TEMED 7uL

 

Note: above volumes are for two gels

-Add 10% APS and TEMED just before pouring gel

-Pour separating gel, ~5mL /gel and add a small volume of t-butanol or 2-propanol and let stand ~15 minutes until gel polymerizes

-After gel polymerizes, pour off t-butano. Remove excess with P200 pepette and paper towel

-Pour stacking gel and let stand until polymerized

 

General Parameters:

SDS-PAGE

  1. aliquot 25- 40ug of protein /20uL in 6x loading buffer boil samples briefly at 100oC
  2. load 20uL of sample per well
  3. use 10uL of Kaleidoscope standard
  4. run gel at 50V through the stack
  5. continue to run gel at 120V (100-150V) TRANSFER to nitrocellulose or PBDF membrane (only if using PBDF membrane, soak in MeOH for a few minutes prior to transfer)
  6. 2 pieces of paper / one membrane / 2 sponges -assemble such that paper dark side of plastic holder is @ (-) end and clear side is at (+) end and be consistent since current will travel (-) to (+) place membrane on (+) end with paper surrounding and then sponges
  7. run @ 100V for 1 hour INCUBATIONS -block in 5% milk for 45min to 1hr
  8. rince membrane in TBST 2x quick then 2x for 5 minutes each rinse
  9. add 1 antibody and let incubate overnight
  10. wash membrane 2x TBST quick then one 5 minute rinse
  11. add secondary antibody in 5% milk and incubate for .5 to 1 hour -wash 2x quick rinse with TBST then two 5min washes
  12. wash one time TBS for 5min DEVELOP (use Pierce SuperSignal West Pico Chemiluminescent Substrate for detection of HRP product #34080)
  13. add 4mL peroxide solution and 4mL enhancer solution and let stand for one minute -expose to film 30s initially, then adjust to get appropriate exposure STRIP
  14. Add 270uL of BME to 50mL of stripping buffer
  15. Let incubate with gel membrane @ 55C for 30 minutes -rinse 3x with TBST
  16. go to above step (incubation with 1 antibody) note: incubation and wash times may vary as per antibody

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