Buffers

Western Blotting.

Posted Feb 12, 2025 Updated Mar 25, 2025

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3 min read

Buffers

Online calculators for some buffers in the end ….

HEPES

compound	weight [g]	volume [mL; H₂O]	final conc
HEPES	238.3	up to 1 L	1 M
HEPES	119.15	up to 0.5 L	1 M
HEPES	119.15	up to 1 L	0.5 M
HEPES	59.58	up to 0.5 L	0.5 M

HEPES, Mw = 238.3 g/mol

PBS fellowship

10xPBS

compound	Mw [g/mol]	weight [g]/1L	final conc
Na₂HPO₂*2H₂O	177.99	17.99	100 mM
K₂HPO₄	136.09	2.45	18 mM
NaCl	58.443	80.067	1.37 M
KCl	74.55	2.013	27 mM

Note: 10x PBS is offten available commercial as a tables, liquid etc.

PBS tween (PBS-T)

compound	stock conc	final conc	volume
PBS	10x	1x	100 mL
Tween20		0.1%	1 mL
H₂O			up to 1 L

10xTBS (Tris-buffered saline buffer)

compound	Mw [g/mol]	weight [g]/1L	final conc
Tris base	121.1	24	mM
NaCl	58.4	88	mM
H₂O			up to 1 L
adjust pH to 7.6

10xTBS alternative recipe

combines Tris base and Tris-HCl. This avoids the large volume of potentially hazardous hydrochloric acid that is needed to neutralize a solution of Tris base alone

compound	Mw [g/mol]	weight [g]/1L	final conc
Tris HCl	157.6	24	mM
Tris base	121.1	5.6	mM
NaCl	58.4	88	mM
H₂O			up to 1 L
adjust pH to 7.6

TBS tween (TBS-T)

compound	stock conc	final conc	volume
TBS	10x	1x	100 mL
Tween20		0.1%	1 mL
H₂O			up to 1 L

PBS casein (PBSC)

compound	stock conc	final conc	volume
PBS	10x	1x	5 mL
casein	10x	1x	5 mL
	or 1g / 100 mL buffer
H₂O			up to 50 mL

casein is vey difficult to solubilize. You can do it with a magnetic stirrer. If casein is slowly added to the buffer while stirring you can solubilize it.
alternatively: casein resuspended in an alkaline solution and then the neutralization and addition of NaCl results in a casein solution in TBS. The key is to neutralize with HCl very gradually. Good luck!
skimmed milk contains a lot of casein and is easily solubilized

PBSC tween (PBSC-T)

compound	stock conc	final conc	volume
PBS	10x	1x	5 mL
casein	10x	1x	5 mL
	or 1g / 100 mL buffer
Tween20		0.1%	1 mL
H₂O			up to 50 mL

Western blot

10x blotting buffer

we use it for Biorad wet transfer

compound	Mw [g/mol]	weight [g]/1L	final conc
TrisBase	121.4	30.3	250 mM
glycine	75.07	144.1	2 M

1x blotting buffer

compound	volume [mL]	final conc
10x blotting buffer	200	1x
MeOH	400	20%
H₂O	1400	(up to 2000)

SDS Sample Buffer

compound	Mw [g/mol]	stock conc	final conc	volume [mL]
LDS	272.33	4x	1x	0.375
DTT	1 M	0.2 M	0.3
H₂O			0.825
total			1.5

Laemly buffer

4x (20 mL)
compound	stock conc		volume/weight
TrisHCl	1 M; pH 6.8		4.8 mL
glycerol			8 mL
SDS			1.6 g
mercaptoethanol
BFB			8 g
H₂O			6.2 mL

BFB …. bromphenol blue
for best results, do not store sample buffer with 2-mercaptoethanol
alternatively, add dithiothreitol (DTT or Cleland’s reagent) to a final concentration of 50 mM
4x Laemmli sample buffer (SB): dilute 3 parts sample with 1 part 4x Laemmli SB; more sample buffer can be added if necessary

IF

cytoskeletal buffer (pre-extraction)

compound	stock conc	final conc	volume [mL]	volume [mL]
HEPES	1 M; pH 7.7	25 mM	0.5	1
NaCl	5 M	50 mM	0.2	0.4
EDTA	0.5 M	1 mM	40 μL	80 μL
MgCl₂	1 M	3 mM	60 μL	120 μL
sucrose	2 M	300 mM	3	6
TritonX	10 %	0.5 %	1	2
H₂O			15.2	30.4
total			20	40

Online calculators and resources

Protocols, Buffers

buffers WB IF FACS

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