11/5/24, 11:37 AM Western blot · Benchling

Western blot
Introduction
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Materials
› RIPA buffer
› NaCl 150mM
› NP-40 1%
› Sodium deoxycholate 1%
› SDS 0.1%
› Tris-HCl pH 7.6 25mM
›

Procedure

Nuclear extraction

1. Centrifuge cells

2. Wash in ice-cold PBS + PI

3. Add ice-cold RIPA lysis buffer to the cell pellet

with PI added

4. 10m ice

5. Bioruptor 4C for 5 mins to increase yield

30s on 30s off x3-5 cycles

6. Centrifuge 14000 rpm 15m @ 4C

7. Collect supernatant and place on ice

after bioruptor, you often do not even see pellet

8. Quantitate by desired method

9. Alternate option is to count cells and do not use RIPA, but just directly boil in Laemmli buffer. You cannot quantitate this
way, but it's faster and if you're using equivalent numbers of cells you should be OK

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RIPA recipe

A B C D E F G

1 Stock conc. Final conc. Vol. in 42.5cc

2 5M NaCl 5 NaCl 150mM 0.15 1.275 mL
3 10% NP-40 0.1 NP-40 1% 0.01 4.250 mL
Sodium
4 deoxycholate 0.01 0.425 g
1%
5 10% SDS 0.1 SDS 0.1% 0.001 0.425 mL
1M Trizma Tris-HCl pH 7.6
6 1 0.025 1.063 mL
supelco 1L 25mM
7 H2O 35.5 mL
8 42.5

Run gel

10. Load protein

Load 5ug total protein for histones

Typically you load 10ug
Can load more if you have it (sometimes it’s better)
Precast gels come in different sizes. Large wells hold up to 30ul, small up to 20ul.
Calculate how much you want to load, mix with 1x Laemmli loading buffer, and boil for 10 min @100C
4x Laemmli is around if you need it (for dilute samples)

11. Bio-Rad precision plus Protein dual color standards: 10ul for mini/midi gel electrophoresis

12. Westerns with Novex pre-cast gels

13. Set-up gel apparatus with precast gels (remove tape and comb)

14. Fill inner and outer (~1/3 height of the gel) chambers with 1X MES running buffer

15. Pipette gently to flush/wash out wells

16. Load gel with thin pipette loading tips

17. Run gel at constant voltage ~125V (higher V increases temp, so add more buffer to the outer chamber to help cool the
gel)

18. Stop voltage when the pre-stained ladder has migrated the desired distance or dye front is off the gel

Transfer

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11/5/24, 11:37 AM Western blot · Benchling

19. Transfer with PVDF membrane

20. Have pre-cooled (stored in 4C) transfer buffer ready when setting up transfer

21. 1X Novex Transfer Buffer with 10% MeOH (50ml 20X Transfer Buffer, 100ml MeOH, and 850ml dH2O)

22. For each gel you need:

PVDF membrane cut to the size of the gel

2 sheets of filter paper cut to the size of the gel
~4 sponges
1 transfer box (do not overlap gel/membrane sandwiches within a transfer box)

23. Open gel plates, cut-off undesired parts and transfer gel into a dish with transfer buffer

24. Activate the PVDF by briefly soaking in MeOH, pour off MeOH (can be reused), and replace with Transfer buffer

25. Soak sponges in dish with transfer buffer

26. Make transfer sandwich

Starting with deep part of the Transfer box (w/ negative terminal) lying on the bench add in the following order:
2 sponges soaked in transfer buffer
1 pre-wet filter paper (can slip the filter paper under the gel while it’s floating in transfer buffer. This allows you to pick
up gel with filter paper and avoid tears)
protein gel
PVDF membrane
1 pre-wet filter paper
2 sponges soaked in transfer buffer
Transfer box lid (w/ positive terminal)
(Can reuse all of the transfer buffer not put in the buffer tank)

27. Put assembled transfer case in a buffer tank

28. Fill outer tank with cold H2O to maintain low temp

29. Run transfer in cold room at constant voltage of 30V for 1h

30. Disassemble transfer set-up (prestained ladder bands should be on membrane)

Stain

31. Block membrane in PBST w/ 5% dry milk, rock at RT for ~30 min

32. Incubate primary antibody O/N at 4C w/rocking

33. Wash 3x with PBST, rocking at RT for ~5min per wash

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11/5/24, 11:37 AM Western blot · Benchling

34. Incubate with secondary HRP-conjugated antibody with rocking at RT for ~1h (1:10,000 for HRP Rb)

35. Wash 3x with PBST, rocking at RT for ~5min per wash

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